CN110944665B - AMHRII binding compounds for preventing or treating lung cancer - Google Patents

Abstract

The present invention relates to the use of a human AMHRII binding agent for the prevention or treatment of lung cancer, in particular to the use of a human AMHRII binding agent for the prevention or treatment of non-small cell lung cancer (NSCLC), even more particularly to the use of a human AMHRII binding agent for the prevention or treatment of NSCLC selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC, and neuroendocrine NSCLC.

Description

AMHRII binding compounds for preventing or treating lung cancer

Technical Field

The present invention relates to the field of lung cancer treatment.

Background Art

Lung cancer is a malignant transformation and expansion of lung tissue and causes 1.3 million deaths worldwide each year. It is the most common cause of cancer-related death in men and the second most common cause in women.

The World Health Organization classifies lung cancer into four major histological types: (1) squamous cell carcinoma (SCC), (2) adenocarcinoma, (3) large cell carcinoma, and (4) small cell lung cancer (SCLC). The term non-small cell lung cancer (NSCLC) includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.

Non-small cell lung cancer (NSCLC) is grouped together because their prognosis and treatment are roughly the same. There are three main subtypes: squamous cell lung cancer, adenocarcinoma, and large cell lung cancer. Squamous cell carcinoma, which accounts for 29% of lung cancers, also starts in the larger bronchi, but grows more slowly. The size of these tumors varies depending on the diagnosis. Adenocarcinoma is the most common subtype of NSCLC, accounting for 32% of lung cancers. It is the form that starts near the gas exchange surface of the lungs. Most adenocarcinoma cases are related to smoking. However, in people who have never smoked ("never smokers"), adenocarcinoma is the most common form of lung cancer. Bronchioloalveolar carcinoma is a subtype of adenocarcinoma that is more common in female never smokers and may respond differently to treatment. Other subtypes of NSCLC are neuroendocrine lung cancer (NE), alveolar lung cancer (AT), and large cell carcinoma (a fast-growing form), which accounts for 9% of lung cancers that grow near the surface of the lung.

Small cell lung cancer (SCLC, also called "oat cell carcinoma") is a less common form of lung cancer. It tends to start in the larger respiratory tract and grows rapidly to a large size. The oncogene most often involved is L-myc. The "oat" cells contain dense neurosecretory granules, giving them an endocrine/paraneoplastic syndrome association. It is initially more sensitive to chemotherapy, but ultimately has a poorer prognosis and is often metastatic by the time it appears. This type of lung cancer is strongly associated with smoking.

Other types of lung cancer include carcinoid, adenoid cystic carcinoma (cylindroma), and mucoepidermoid carcinoma.

Early detection is difficult because clinical symptoms usually do not appear until the disease is advanced. Currently, diagnosis is aided by the use of chest x-rays, analysis of the types of cells contained in saliva, and fiber optic examination of bronchial passages. Treatment options are determined by the type and stage of the cancer and include surgery, radiation therapy, and/or chemotherapy. Although much research has been done on therapies for the disease, lung cancer remains difficult to treat.

Known treatments for lung cancer include surgery, chemotherapy, radiation therapy, and targeted drug therapy.

Targeted therapies, especially targeted immunotherapies, have the potential to benefit lung cancer patients who do not respond to more conventional chemotherapy or radiation therapy. Targeted immunotherapies include the use of monoclonal antibodies.

The monoclonal antibodies bevacizumab (anti-VEGF antibody) and ramucirumab (anti-VEGFR2 antibody) are designed to prevent tumors from generating new blood vessels, while necitumomab (anti-EGFR) targets growth by preventing the action of another growth factor. Currently, at least two immune checkpoint inhibitors (pembrolizumab/anti-PD1 and nivolumab/anti-PD1) targeting antibodies are approved for patients with lung cancer. Today, long-term remissions and longer survival rates can be achieved using such immune-based treatments, such as checkpoint inhibitors, monoclonal antibodies, therapeutic vaccines, and adoptive cell therapy.

However, there remains a need in the art for additional tools for lung cancer therapy, which may be an alternative or supplement to existing therapies.

Summary of the invention

The present invention relates to the use of human AMHRII binding agents for preventing or treating lung cancer. Then, the present invention relates to the use of human AMHRII binding agents in a method for preventing or treating lung cancer in a patient suffering from lung cancer.

The lung cancer may be selected from the group comprising non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC.

In a preferred embodiment, the human AMHRII binding agent is used to treat the above-specified lung cancers that express AMHRII at a sufficient expression level at the cell membrane.

In a most preferred embodiment, the sufficient expression level is expressed as a threshold AMHRII expression score value as described in detail elsewhere in this specification.

In some embodiments, the human AMHRII binding agent consists of an anti-AMHRII monoclonal antibody.

In some embodiments, the human AMHRII binding agent is composed of an antibody drug conjugate (ADC).

In some embodiments, the human AMHRII binding agent consists of an AMHRII binding engineered receptor.

In some embodiments, the human AMHRII-binding agent is composed of cells expressing AMHRII-binding engineered receptors (such as CAR T cells or NK T cells expressing AMHRII-binding engineered receptors).

In some embodiments, the AMHRII binding agent is combined with one or more different anti-cancer agents.

The present invention also relates to a method for determining whether an individual is suitable for lung cancer treatment using an AMHRII binding agent as defined above, wherein the method comprises the step of determining whether a lung tumor tissue sample previously obtained from the individual expresses AMHRII protein at the cell surface.

The present invention relates to a method for determining whether an individual is responsive to lung cancer treatment using an AMHRII binding agent as defined above, wherein the method comprises the step of determining whether a lung tumor tissue sample previously obtained from the individual expresses AMHRII protein at the cell surface.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the amino acid sequences of the VH domain and VL domain of multiple variants of the 3C23 monoclonal antibody. Figure 1A shows the VH domain of each antibody variant. Figure 1B shows the VL domain of each antibody variant.

FIG. 2 shows AMHRII expression by various cancer cell lines.

Figure 2A shows AMHRII mRNA expression of cancer cell lines. Abscissa: from left to right of Figure 2A: HCT116 (colon colorectal cancer), COV434-WT (human ovarian granulosa cell tumor), K562 (human myeloid leukemia) and OV90 (human malignant papillary serous adenocarcinoma). Ordinal axis: AMHRII mRNA expression levels as determined by RT-qPCR, expressed in arbitrary units (RQ).

Figures 2B to 2F: Membrane expression of AMHRII protein by the same cancer cell lines as Figure 2A: HCT116 (Figure 2B), COV434-WT (Figure 2C), K562 (Figure 2D), NCI-H295R (Figure 2E) and OV90 (Figure 2F). Abscissa: Fluorescence signal intensity (FL2-A dye) expressed in arbitrary units. Ordinal axis: Cell count.

FIG. 3 shows AMHRII expression by various lung cancer cells as measured by flow cytometry.

Fig. 3A shows the AMHRII protein membrane expression of the cell (Ref Lu7860) of the xenograft from squamous cell lung cancer patient source. Fig. 3B shows the AMHRII protein membrane expression of the cell (Ref Lu7166) of the xenograft from large cell lung cancer patient source. Fig. 3C shows the AMHRII protein membrane expression of the cell (RefLu7298) of the xenograft from squamous cell lung cancer patient source. Fig. 3D shows the AMHRII protein membrane expression of the cell (Ref Lu7414) of the xenograft from squamous cell lung cancer patient source. Fig. 3E shows the AMHRII protein membrane expression of the cell (Ref Lu7558) of the xenograft from pleomorphic cell lung cancer patient source. Abscissa: fluorescence intensity (FL2-A dye), expressed in arbitrary units. Ordinal axis: cell count.

FIG. 3F shows membrane expression of AMHRII protein by cells from the healthy margin of surgically resected human NSCLC (whose FACS curve is shown in FIG. 3G ).

FIG. 3G shows membrane expression of AMHRII protein by cells from fresh samples of surgically resected human NSCLC.

In Figure 3: (i) left peak: cells incubated with irrelevant isotype antibodies; (ii) right peak: cells incubated with 3C23K anti-AMHRII antibody.

Abscissa: fluorescence intensity (FL2-A dye), expressed in arbitrary units. Vertical: cell count.

Figure 4 shows the relative body weight changes of animals xenografted with human lung cancer cells. Treatment started on the 18th day after SC131 implantation. 20 mg/kg of vehicle and GM102 were administered i.v. once every two weeks for 3 weeks. At D0, 20 mg/kg of docetaxel was administered slowly i.v. once. 5 mg/kg of cisplatin and 100 mg/kg of gemcitabine were administered i.p. once a week for 1 to 3 weeks. Initial group size: 9 animals. Vertical axis: relative body weight, expressed in kg (mean +/- standard error of the mean (sem)). Horizontal axis: ● Vehicle; ■ GM102 20 mg/kg; ▲ Docetaxel 20 mg/kg; ▼ Combination of GM102 and docetaxel; ◆ Combination of cisplatin 5 mg/kg and gemcitabine 100 mg/kg; ○ Combination of GM102, cisplatin and gemcitabine.

Figure 5 shows changes in tumor growth induced by 3C23K anti-AMHRII antibody in combination with or without other anticancer agents in animals xenografted with human lung cancer cells. Treatment began on day 18 after SC131 implantation. 20 mg/kg of vehicle and GM102 were administered iv once every two weeks for 3 weeks. At D0, 20 mg/kg of docetaxel was slowly administered iv once. 5 mg/kg of cisplatin and 100 mg/kg of gemcitabine were administered ip once weekly for 1 to 3 weeks. Initial group size: 9 animals. Ordinal axis: tumor volume, expressed in mm ³ (mean +/- sem). Abscissa: ● Vehicle; ■ GM102 20 mg/kg; ▲ Docetaxel 20 mg/kg; ▼ Combination of GM102 and docetaxel; ◆ Combination of cisplatin 5 mg/kg and gemcitabine 100 mg/kg; ○ Combination of GM102, cisplatin and gemcitabine.

Figure 6 shows the anti-tumor activity of 3C23K anti-AMHRII antibodies in combination with or without other anti-cancer agents in animals xenografted with human lung cancer cells. Treatment started on day 18 after SC131 implantation. 20 mg/kg of vehicle and GM102 were administered i.v. once every two weeks for 3 weeks. At D0, 20 mg/kg of docetaxel was administered slowly i.v. once. 5 mg/kg of cisplatin and 100 mg/kg of gemcitabine were administered i.p. once a week for 1 to 3 weeks. Initial group size: 9 animals. Vertical axis: TC as expressed in percentage. Horizontal axis: ● Vehicle; ■ GM102 20 mg/kg; ▲ Docetaxel 20 mg/kg; ▼ Combination of GM102 and docetaxel; ◆ Combination of cisplatin 5 mg/kg and gemcitabine 100 mg/kg; ○ Combination of GM102, cisplatin and gemcitabine.

FIG. 7 shows changes in tumor growth induced by GM102 (low-fucose anti-AMHRII antibody) on animals xenografted with squamous non-small cell lung cancer tumor xenografts. Each dashed curve: xenografted animals administered vehicle solution. Each continuous curve: xenografted animals administered GM102. Abscissa: time period after treatment start, expressed in days. Ordinal axis: tumor volume, expressed in mm ³ .

Figure 8 shows changes in tumor growth induced by GM102 (low-fucose anti-AMHRII antibody) on animals xenografted with squamous non-small cell lung cancer tumor xenografts at day 28 after the start of treatment. Figures 8A and 8B, ordinate: tumor volume, expressed in mm ^3. Figures 8A and 8B, abscissa: (i) left: xenografted animals administered vehicle solution; (ii) right: xenografted animals administered GM102. Figure 8A: Absolute results for each xenografted animal tested. Figure 8B: Mean +/- standard deviation calculated from the results depicted in Figure 8A.

DETAILED DESCRIPTION

The inventors surprisingly showed that the AMHRII receptor is expressed at the cell membrane of non-small cell lung cancer tissue, especially in epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, pleomorphic cell carcinoma NSCLC, squamous cell carcinoma NSCLC and neuroendocrine NSCLC subtypes. In contrast, no membrane level of AMHRII was detected in SCLC or NSCLC from neuroendocrine subtypes or acinar subtypes.

The term "AMHR-II" means human anti-Mullerian hormone type II receptor. The sequence of human AMHR-II is described herein as SEQ ID NO. 18 (lacking the signal peptide MLGSLGLWALLPTAVEA (SEQ ID NO: 17)).

As used herein, the term "PDX" is an acronym for "patient-derived xenograft." Patient-derived xenografts are highly used in vivo models of cancer in which tissue or cells from a patient's tumor are implanted (i.e., "transplanted") into an immunodeficient non-human mammal (e.g., an immunodeficient mouse).

As shown in the Examples herein, the inventors discovered that AMHRII is expressed at the cell membrane of lung cancer tissue at variable frequencies depending on the lung cancer subtype in question.

To the best of the inventors' knowledge, membrane expression of AMHRII in lung cancer cells is shown herein for the first time.

Illustratively, as shown in the examples herein, AMHRII is expressed more frequently by cancer cells derived from tumor tissue of patients with epidermoid NSCLC lung cancer or adenocarcinoma NSCLC large cell NSCLC lung cancer than by cancer cells derived from tumor tissue of patients with squamous NSCLC or large cell NSCLC. Detecting a relatively high frequency means that cancer patients with one of these four lung cancers are more frequently suitable for (i.e., more frequently respond to) anticancer treatments targeting AMHRII, but such anticancer treatments are less frequently associated with treating patients with neuroendocrine NSCLC.

As shown in the examples herein, any NSCLC lung cancer can be treated by an AMHRII binding agent, provided that tumor cells from the non-gynecological tumor express AMHRII at their membrane, and therefore provided that the presence of AMHRII protein at the tumor cell membrane can be detected or determined according to any method.

Thus, the experimental data presented in the examples herein demonstrate that the same AMHRII binder (here, an anti-AMHRII monoclonal antibody) is effective for treating a variety of different NSCLC lung cancers, provided that the AMHRII target protein is expressed at the tumor cell membrane.

Incidentally, in the field of anticancer active ingredients consisting of target binding molecules (e.g., target binding antibodies), it is not unprecedented that the same active ingredient is effective for treating multiple different cancers. Illustratively, an anti-PD1 antibody named pembrolizumab has been authorized by the U.S. Food and Drug Administration (FDA) as an active ingredient that can be used to treat multiple different types of cancer, provided that the cancers share the same physiological characteristics.

As used herein, expressing AMHRII at the cell membrane of a lung cancer cell means that the lung cancer cell expresses AMHRII at a given quantifiable level or above the quantifiable level.

According to some embodiments, by determining whether lung cancer cells from a sample previously collected from an individual with lung cancer express AMHRII at their membrane, the responsiveness of the individual to treatment with an AMHRII binding molecule can be assessed.

According to some embodiments, by determining whether lung cancer cells from a sample previously collected from an individual with lung cancer express AMHRII at their membrane above a determined threshold, the responsiveness of the individual to treatment with an AMHRII binding molecule can be assessed.

The level of AMHRII membrane expression, which can be used in some embodiments to determine the responsiveness of patients with non-gynecological cancers to treatment with an AMHRII binder (e.g., an anti-AMHRII antibody), can be assessed using a variety of techniques, including (i) the percentage of tumor cells contained in a tumor sample that express AMHRII at their membrane, (ii) the average amount of AMHRII protein at the tumor cell membrane, and (iii) a FACS AMHRII signal plot of tumor cells contained in the tested tumor cell sample.

According to some embodiments, lung cancer cells contained in a tumor sample previously collected from an individual with lung cancer may be assessed as expressing membranous AMHRII when membranous AMHRII is detected in 5% or more of lung tumor cells contained in the tumor sample.

Thus, in some embodiments, when 5% or more of lung tumor cells contained in a tumor sample previously collected from an individual with lung cancer express AMHRII at their membrane, the individual is determined to be responsive to treatment with an AMHRII binding agent.

Methods for determining the frequency (eg, percentage) of tumor cells expressing membrane AMHRII protein are disclosed elsewhere in this specification, including in the Examples herein.

According to some embodiments, by determining the average amount of AMHRII protein present at tumor cell membranes contained in tumor samples previously collected from patients with lung cancer, the responsiveness of the patient to cancer treatment with an AMHRII binding agent (e.g., an anti-AMHRII antibody) can be assessed.

In some embodiments, when the average number of membrane AMHRII proteins expressed by tumor cells contained in a tumor sample previously collected from a patient with lung cancer is 10,000 AMHRII proteins or more, the patient can be classified as responsive to treatment with an AMHRII binding agent, such as anti-AMHRII antibody treatment.

The evaluation of the quantity of AMHRII protein expressed at the membrane of lung tumor cells can be performed by using conventional methods, the conventional methods comprising: (a) a step of incubating a sample containing cells from a tumor tissue sample previously collected from a patient with a detectable compound (such as a fluorescently labeled anti-AMHRII antibody) that specifically binds to the AMHRII protein, and additionally (b) a step of determining the quantity of the detectable compound (e.g., the quantity of fluorescently labeled anti-AMHRII antibodies) bound to each test cell from the sample. As shown in the examples herein, the quantity of AMHRII protein expressed at the membrane of tumor cells can be evaluated, for example, by using well-known fluorescence activated cell sorting (FACS) technology.

In yet other embodiments, by analyzing the AMHRII FACS profile of tumor cells contained in a tumor sample previously collected from a patient with lung cancer, the patient can be classified as responsive to treatment with an AMHRII binding agent, such as anti-AMHRII antibody treatment.

According to these other embodiments, in a fluorescence activated cell sorting (FACS) method, a patient with lung cancer can be classified as responsive to treatment with an AMHRII binder, for example, as responsive to treatment with an anti-AMHRII antibody, when the ratio of the mean fluorescence intensity (MFI) value obtained from (i) tumor cells incubated with an isotype fluorescently labeled antibody to (ii) the mean fluorescence intensity of tumor cells incubated with an anti-AMHRII fluorescently labeled antibody is 1.5 or greater.

To determine the mean fluorescence intensity ratio, both the isotype antibody and the anti-AMHRII antibody are labeled with the same fluorescent agent (such as the Alexa Fluor 488 dye sold by the Company ThermoFisher Scientific), as shown in the examples herein.

In some additional embodiments, the responsiveness of a lung cancer individual to treatment with an AMHRII binder can be determined by calculating an AMHRII expression score that allows for the differentiation of (i) lung cancer cells expressing membrane AMHRII from lung cancer that can be treated with an AMHRII binder and (ii) lung cancer cells expressing membrane AMHRII from lung cancer that cannot be treated with an AMHRII binder.

Thus, the inventors have determined that patients with lung cancer who are particularly suitable for cancer treatment using the AMHRII binding agents described herein (i.e., patients with lung cancer who are particularly responsive to cancer treatment using the AMHRII binding agents described herein) include those patients who have cancer tumors that express AMHRII at the cell membrane at high levels sufficient to constitute relevant cellular targets to be destroyed.

Then, according to these further embodiments, the inventors have determined that a minimum AMHRII expression level measured in a cancer cell sample from a lung cancer patient can confirm that the patient is responsive to treatment with an AMHRII binding agent, and thus the patient can be treated with an AMHRII binding agent described herein.

Therefore, when the level of AMHRII expression in lung cancer cells contained in a sample previously collected from an individual with lung cancer is assessed by determining (i) the frequency of tumor cells expressing membrane AMHRII (e.g., the percentage of tumor cells expressing AMHRII at their membrane) and (ii) the level of AMHRII membrane expression by said tumor cells (e.g., the average amount of membrane AMHRII protein per cell), the responsiveness of said individual to treatment with an AMHRII binding agent can also be determined.

Therefore, in some of these additional embodiments, the inventors have determined that the responsiveness of a lung cancer patient to a human AMHRII binder, such as an anti-human AMHRII antibody, requires that in a sample of tumor cells previously collected from the patient, (i) the tumor cells contained in the sample have a minimum average number of human AMHRII proteins at their membranes and (ii) the frequency of cells expressing human AMHRII at their membranes (e.g., the percentage of cells expressing human AMHRII at their membranes) is at least a threshold.

Therefore, another method is also described herein that can also be used to determine a specific AMHRII expression score value to allow for the distinction between (i) lung cancer patients who are not suitable for cancer treatment using an AMHRII binder (i.e., lung cancer patients who do not respond to cancer treatment using an AMHRII binder) and (ii) lung cancer patients who are suitable for cancer treatment using an AMHRII binder (i.e., lung cancer patients who respond to cancer treatment using an AMHRII binder (e.g., an anti-human AMHRII antibody)).

More precisely, according to the above method embodiments, patients suffering from lung cancer as described herein and who can be treated with the AMHRII binding agent as described herein for lung cancer are preferably those whose membrane AMHRII expression score value has been determined to be 1.0 or greater.

The membrane AMHRII expression score can be based on the immunohistochemical evaluation of AMHRII expression of the tested lung cancer cells and is the average of the membrane AMHRII scores determined from multiple lung cancer cell samples derived from different individuals with lung cancer, and wherein, (i) if no AMHRII expression is detected, the individual membrane AMHRII score of the given lung cancer cell sample is assigned to 0, (ii) if significant AMHRII expression is detected, the individual membrane AMHRII score of the given lung cancer cell sample is assigned to 1, (iii) if high AMHRII expression is detected, the individual membrane AMHRII score of the given lung cancer cell sample is assigned to 2, and (iv) if overexpression of AMHRII is detected, the individual membrane AMHRII score of the given lung cancer cell sample is assigned to 3.

In fact, there is a relationship between (i) the score assigned to the membrane AMHRII expression level by the above-mentioned immunohistochemical evaluation and (ii) the average amount of AMHRII protein expressed by each lung cancer cell. In the examples herein, it is shown that, starting from a sample of lung tumor cells previously collected from lung cancer patients, by determining the average amount of membrane AMHRII protein per cell, it is also possible to evaluate the membrane AMHRII expression level that enables the assignment of an individual membrane AMHRII score.

According to the above embodiment of determining the responsiveness of an individual with lung cancer to treatment with an AMHRII binding agent (i.e., to treatment with an anti-AMHRII antibody), for a given lung cancer cell sample, a membrane AMHRII expression score is determined by considering the following two: (i) the frequency of AMHRII expressing cells in the lung cancer cell sample and (ii) the level of membrane AMHRII expression of the AMHRII expressing cells. Typically, the membrane AMHRII expression score of a given lung cancer cell sample is determined by the following formula (I):

E score = frequency x AMHRII_level, where

-E score refers to the membrane AMHRII expression score value of a given lung cancer cell sample,

- frequency means the frequency of cells contained in said lung cancer cell sample in which membrane AMHRII expression is detected, and

-AMHRII_level means the level of AMHRII membrane expression of AMHRII expressing cells contained in the given lung cancer cell sample.

Illustratively, the E-score for a given lung cancer cell sample is determined to be 1.0, where (i) 50% of the cells express AMHRII (frequency value of 0.5), and (ii) the AMHRII expression level (AMHRII_level) is 2.

In some embodiments, the AMHRII expression score (or E score) is determined by immunohistological methods as shown in the examples herein. According to these preferred embodiments, AMHRII membrane expression is assessed by using a detectable antibody specific for AMHRII and by (i) determining the frequency of cells bound to the anti-AMHRII antibody and (ii) determining the intensity of the signal generated by the detectable anti-AMHRII antibody after binding to membrane-expressed AMHRII.

However, as shown in the examples herein, AMHRII-expressing lung cancer cells having a membrane AMHRII expression score of 1.0 or greater have been identified for various lung cancers, albeit at different frequencies.

To determine the level of AMHRII membrane expression, detection of AMHRII at the cell membrane should most preferably be performed by using an anti-AMHRII monoclonal antibody with high affinity and high specificity for AMHRII, which is exemplified by the 3C23K anti-AMHRII monoclonal antibody.

In addition, considering determining AMHRII score, determining AMHRII expression by immunohistochemical method most preferably involves carefully pre-treating the sample before making lung tissue sample contact with appropriate detection reagent (for example high affinity anti-AMHRII monoclonal antibody, such as monoclonal 3C23K antibody, for being bound to AMHRII has the Kd value of 55.3pM). Sample pre-treatment should allow to increase the utilization rate of the detection reagent of the AMHRII molecule expressed at the cell surface. Illustratively, as shown in the example of this paper, pre-treatment method can include the appropriate combination of such as following specific steps: (i) by being exposed to high temperature dewaxing of microwave source and (ii) for amplifying the system of signal produced by the combination of AMHRII binding reagent (such as biotinylated anti-AMHRII antibody) of the detectable reagent compound that can be conjugated with streptavidin subsequently. Due to previous tissue fixation step, pre-treatment dewaxing step seems to be important for reversing the detection signal extinction effect. The inventor has shown that AMHRII detectability is particularly sensitive to the effect of formalin for tissue fixation step.

This means that, although AMHRII binding agents may be relevant therapeutic agents for treating patients with lung cancer, it is preferable to first test tumor-derived lung cancer cells for AMHRII expression to determine whether a particular patient will be administered an AMHRII binding agent as described herein.

Furthermore, the inventors have shown that anti-AMHRII antibodies can be advantageously used in the treatment of lung cancer.

Thus, the inventors have shown herein that agents targeting AMHRII can be used as a novel therapeutic tool for preventing or treating these types of cancer, in particular NSCLC selected from the group comprising epidermoid NSCLC, pleomorphic cell carcinoma NSCLC, and neuroendocrine NSCLC adenocarcinoma NSCLC, large cell NSCLC, and squamous cell carcinoma NSCLC, and neuroendocrine NSCLC.

According to the present invention, the expression "including" such as in "including the following steps" is also understood as "consisting of the following", such as "consisting of the following steps" is also understood as "consisting of the following", such as "consisting of the following steps".

AMH receptor (AMHR or AMHR2 or AMHRII) is a serine/threonine kinase with a single transmembrane domain, belonging to the II type receptor family of TGF-β associated proteins. II type receptor itself binds to ligands, but it is necessary to have type I receptors for signal transduction. Imbeaud et al. (1995, Nature Genet, Vol. 11: 382-388) cloned human AMH II type receptor gene. Human AMH receptor protein is composed of 573 amino acids: 17 amino acids, 127 amino acids, 26 amino acids and 403 amino acids in these 573 amino acids form signal sequence, extracellular domain (ECD), transmembrane domain and intracellular domain (containing serine/threonine kinase domain) respectively.

As used herein, the term "AMHRII" refers to the human anti-Mullerian hormone type II receptor having the amino acid sequence of SEQ ID NO.17.

The expression of anti-Mullerian hormone receptor (AMHRII) has been described in the art in gynecological cancers (i.e., tumors that are mostly infiltrated by immune myeloid cells). AMHRII has been identified as a target molecule for the treatment of gynecological cancers. Antibodies against AMHRII have been produced as therapeutic tools for the treatment of these cancers. The 12G4 anti-AMHRII antibodies and variants thereof for the treatment of ovarian cancer described in PCT Application No. WO 2008/053330 and No. WO 2011/141653, and the 3C23K anti-AMHRII antibodies described in the PCT application can be particularly cited. PCT Application No. WO 2017/025458 can also be mentioned, which discloses a specific therapeutic strategy for ovarian cancer by using anti-AMHRII antibody drug conjugates.

Beck et al. (2016, Cell Reports, Vol. 16: 657-671) also describe the expression of anti-Mullerian hormone receptor gene (AMHRII gene). These authors show that AMH signaling is an important factor for epithelial plasticity, survival signaling and selective drug resistance in NSCLC. Beck et al. (2016) work provides insights into the intracellular mechanism of NSCLC pathogenesis, especially through reports, by using siRNA to regulate various gene expressions of interest, identify and characterize the previously undefined autocrine signaling axis (involving anti-Mullerian hormone and its type II receptor) in NSCLC tumor subgroups. The response to Hsp90 inhibitor genitinib and approved chemotherapy cisplatin is essential. These authors also found low-abundance AMH protein and AMHR2 protein in cells (i.e. A549 and H1299) of three cell lines by Western blotting experiments, and the production of these proteins is blocked by SiRNA targeting corresponding genes.

The inventors have now surprisingly found that AMHRII is also expressed at the surface of various human lung cancer cells, including in particular non-small cell lung cancer (NSCLC) cells, even more in particular NSCLC selected from the group comprising epidermoid NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC and neuroendocrine NSCLC. The inventors have also shown that there is no relationship between (i) AMHRII gene expression of cancer cells and (ii) cell membrane AMHRII protein expression of the same cancer cells.

The inventors' discovery of AMHRII surface expression of human lung cancer cells comes in particular from immunohistochemical tests using anti-AMHRII antibodies, which are performed using human lung tumor tissue samples previously obtained from lung cancer patients. The inventors' discovery of AMHRII surface expression of human lung cancer cells is also obtained from immunohistochemical tests using anti-AMHRII antibodies, which are performed in mice on lung tumor tissue samples derived from human primary lung cancer cell xenografts.

The inventors have also shown that anti-AMHRII antibodies can be used to treat human lung cancers expressing AMHRII at the surface of tumor cells, especially those AMHRII-expressing lung cancers disclosed in this specification, including non-small cell lung cancers, especially epidermoid NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC and neuroendocrine NSCLC. It is worth noting that anti-AMHRII antibodies and anti-AMHRII antibodies in combination with chemical anticancer agents (such as the well-known anticancer agents docetaxel, cisplatin and/or gemcitabine) have shown good anticancer activity.

The inventors have shown that anti-AMHRII antibodies that have been demonstrated in the art to have anti-tumor efficacy against gynecological cancers expressing AMHRII can also be used to prevent or treat lung cancers expressing AMHRII, and in particular those lung cancers expressing AMHRII disclosed in the present specification, such as non-small cell lung cancer, in particular epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC.

More specifically, the examples herein show that an anti-AMHRII antibody named 3C23K exerts anti-tumor activity in vivo against human lung cancer, particularly against non-small cell lung cancer disclosed herein, including when treatment with the anti-AMHRII antibody is combined with treatment with one or more different anti-cancer agents (such as docetaxel, cisplatin and/or gemcitabine).

Furthermore, the present inventors have also shown that the anti-AMHRII 3C23K antibody does not induce detectable toxic events in vivo, in particular, does not induce significant body weight loss.

Therefore, the present invention relates to the use of human AMHRII binding agents for preventing or treating lung cancer, in particular non-small cell lung cancer (NSCLC), more particularly non-small cell lung cancer (NSCLC) selected from the group consisting of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, pleomorphic cell carcinoma and neuroendocrine NSCLC.

The present invention also relates to the use of human AMHRII binding agent for the preparation of a medicament for preventing or treating lung cancer, especially lung cancer selected from the group consisting of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC.

The present invention also relates to a method for preventing or treating lung cancer, in particular lung cancer selected from the group consisting of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC, wherein the method comprises the step of administering the AMHRII binder disclosed in the present specification to an individual in need thereof.

The AMHRII binding agent that can be used according to the present invention does not need to simulate the MIS natural ligand activity.Therefore, the AMHRII binding agent that can be used according to the present invention does not need to activate any cell signaling pathway after being bonded to AMHRII.On the contrary, only require the reagent to have the ability combined with AMHRII, because the reagent is specifically used in the activity (such as cytotoxicity inducing entity) of target-induced cytotoxicity, it covers the anti-AMHRII antibody of anti-AMHRII cytotoxic immunoconjugate, ADCC induction or the anti-AMHRII antibody of ADC induction or express the CAR T cell of AMHRII in conjunction with engineered receptor.

AMHRII binders

As used herein, an AMHRII binding agent encompasses any such agent that specifically binds to AMHRII and when presented in an appropriate manner will cause the death of a target cell expressing AMHRII at its surface following binding of the agent to AMHRII expressed on the cell membrane.

The AMHRII binding agents for use in treating lung cancer as described herein may also be referred to herein as "therapeutic AMHRII binding agents."

Typically, an AMHRII binding agent encompasses a protein or nucleic acid that specifically binds to AMHRII.

AMHRII binding proteins mainly cover proteins comprising one or more complementarity determining regions (CDRs) derived from anti-AMHRII antibodies or AMHRII binding fragments of anti-AMHRII antibodies. It should be understood that the AMHRII binding proteins can be expressed as chimeric antigen receptors (CARs) by engineered cells (such as CAR-T cells, NK T cells or CAR macrophages).

AMHRII binding nucleic acids primarily encompass nucleic acid aptamers that are specifically selected for their specific binding properties to AMHRII.

In some preferred embodiments, the AMHRII binding agent is an anti-AMHRII antibody or an AMHRII binding fragment thereof.

In a most preferred embodiment, the AMHRII binding agent is an anti-AMHRII monoclonal antibody or an AMHRII binding fragment thereof.

According to these preferred embodiments, the anti-AMHRII monoclonal antibodies encompass chimeric anti-AMHRII antibodies, humanized anti-AMHRII antibodies and human AMHRII antibodies, as well as AMHRII binding fragments and AMHRII binding derivatives thereof.

Various AMHRII antibodies are known in the art and can be used as AMHRII binding agents according to the present invention. For the purpose of practicing the present invention, those skilled in the art can use the recombinant human anti-AMHRII No. MHH-57 sold by Creative Biolabs for example.

In some embodiments, an anti-AMHRII antibody that can be used according to the present invention is the humanized 12G4 antibody disclosed in PCT Application No. WO 2008/053330.

In some other embodiments, the anti-AMHRII antibody is a humanized antibody described in PCT Application No. WO 2011/141653, which encompasses the 3C23 antibody and variants thereof, including the 3C23K humanized antibody.

In further embodiments, the anti-AMHRII antibody is those described in PCT Application No. WO 2017/025458. According to these additional embodiments, PCT Application No. WO 2017/025458 discloses an AMHRII binding agent in the form of an antibody drug conjugate (ADC), wherein the anti-AMHRII antibody is linked to a cytotoxic agent.

Monoclonal antibodies directed against the Mullerian hormone type II receptor (and its humanized derivatives) have been developed in the art for the treatment of ovarian cancer (see EP 2097453 Bl and US Pat. No. 8,278,423, which are incorporated herein by reference in their entirety).

In the AMHRII binding agent that can be used according to the present invention, those skilled in the art can use monoclonal antibody 12G4 (mAb 12G4) or its chimeric variant or humanized variant, including antibodies derived from drugs or detectable labels to form ADCs. The hybridoma producing mAb12G4 was deposited in the National Collection of Microorganisms (CNCM, Pasteur Institute, 25 Dr. Cruz Avenue, 15th Arrondissement, Paris, France 75724) according to the terms of the Budapest Treaty on September 26, 2006, and the CNCM deposit number is 1-3673. The variable domains of the light chain and heavy chain of mAb 12G4 have been sequenced as the complementary determining regions (CDRs) of mAb 12G4 (see EP 2097453B1 and U.S. Patent No. 8,278,423, which are hereby incorporated by reference in their entirety). mAb 12G4 and its chimeric variant or humanized variant can be used to produce ADCs as disclosed herein.

PCT Application No. PCT/FR2011/050745 (International Publication No. WO/2011/141653) and U.S. Patent No. 9,012,607 (each of which is hereby incorporated by reference in its entirety) disclose novel humanized antibodies derived from mouse 12G4 antibodies. For the purposes of the present invention, these humanized antibodies can be used as AMHRII binders. In the specific embodiments disclosed in PCT Application No. WO/2011/141653, the antibodies are those identified as 3C23 and 3C23K. The nucleic acid sequences and polypeptide sequences of these antibodies are provided herein as SEQ ID NO: 1 to SEQ ID NO: 16. In some aspects of the present invention, the anti-AMHRII antibodies of interest can be referred to as "comprising a light chain comprising SEQ ID NO: and a heavy chain comprising SEQ ID NO:". Therefore, in various embodiments, particularly preferred antibodies (including antibodies for producing ADCs) include:

a) a light chain comprising SEQ ID NO: 2 and a heavy chain comprising SEQ ID NO: 4 (3C23 VL sequence and 3C23 VH sequence without a leader sequence);

b) a light chain comprising SEQ ID NO: 6 and a heavy chain comprising SEQ ID NO: 8 (3C23K VL sequence and 3C23K VH sequence without a leader sequence);

c) a light chain comprising SEQ ID NO: 10 and a heavy chain comprising SEQ ID NO: 12 (3C23 light chain and 3C23 heavy chain without a leader sequence);

d) a light chain comprising SEQ ID NO: 14 and a heavy chain comprising SEQ ID NO: 16 (3C23K light chain and 3C23K heavy chain without a leader sequence).

Other antibodies (e.g., humanized antibodies or chimeric antibodies) can be based on the heavy chain sequences and light chain sequences provided in Figures 1A and 1B (e.g., antibodies, such as humanized antibodies or chimeric antibodies containing CDR sequences disclosed in the figures) can be used as anti-MAHRII binding agents of interest, including antibodies for forming ADCs. Therefore, the present invention also relates to the use of anti-AMHRII antibodies comprising CDRs comprising (or consisting of) the following sequences:

-CDRL-1: RASX1X2VX3X4X5A (SEQ ID NO. 65), wherein X1 and X2 are independently S or P, X3 is R or W or G, X4 is T or D, and X5 is I or T;

-CDRL-2 is PTSSLX6S (SEQ ID NO. 66), wherein X6 is K or E; and

-CDRL-3 is LQWSSYPWT (SEQ ID NO. 67);

-CDRH-1 is KASGYX7FTX8X9HIH (SEQ ID NO. 68), wherein X7 is S or T, X8 is S or G, and X9 is Y or N;

-CDRH-2 is WIYPX10DDSTKYSQKFQG (SEQ ID NO. 69), wherein X10 is G or E; and

-CDRH-3 is GDRFAY (SEQ ID NO. 70).

The present invention also relates to the use of ADCs generated using such anti-AMHRII antibodies for treating lung cancer, especially non-small cell lung cancer and small cell lung cancer.

Antibodies (e.g., chimeric antibodies or humanized antibodies) within the scope of the present application include those disclosed in the following table: Alternatively, human monoclonal antibodies that specifically bind to AMHR-II can be used to prepare ADCs. The 3C23K antibody is defined by:

- SEQ ID NO: 19 for the VH amino acid sequence,

- SEQ ID NO: 36 for the VL amino acid sequence.

Table 1 below lists anti-AMHRII humanized antibodies that can be used according to the present invention.

Table 1: Anti-AMHRII Antibodies

Anti-AMHRII antibodies, AMHRII binding fragments or AMHRII binding derivatives of anti-AMHRII antibodies

The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (see below) so long as they exhibit the desired biological activity.

Therefore, as used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, by way of example, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules (such as shark immunoglobulins) produced during immune responses in any vertebrate (e.g., mammals, such as humans, goats, rabbits, and mice, and non-mammalian species). Unless otherwise specifically stated, the term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to AMHRII, substantially excluding binding to other molecules (i.e., molecules unrelated to AMHRII). The term "antibody" also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, 111.); Kuby, J., Immunology, 7th Edition, W.H. Freeman & Co., New York, 2013.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody including the population is identical except for a possible naturally occurring mutation that may be present in a small amount. Monoclonal antibodies are highly specific for a single antigen. In addition, in contrast to the polyclonal antibody preparation that generally includes different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. The modifier "monoclonal" should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibody to be used according to the present invention can be prepared by the hybridoma method described by Kohler et al., Nature 256:495 (1975), or can be prepared by a recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibody" can also be separated from a phage antibody library using, for example, Clackson et al., Nature 352:624-628 (1991) or Marks et al., J.MoI Biol.222:581-597 (1991) described technology.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed by antibody fragments.

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation, kappa light chains and lambda light chains referring to the two major antibody light chain isotypes.

As used herein, the term "complementarity determining region" or "CDR" refers to the part of the two variable chains (heavy chain and light chain) of an antibody that recognizes and binds to a specific antigen. CDR is the most variable part of the variable chain and provides the antibody with its specificity. There are three CDRs on each of the variable heavy (VH) chain and the variable light (VL) chain, so there are a total of six CDRs per antibody molecule. CDR is primarily responsible for binding to the epitope of the antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, which are numbered sequentially from the N-terminus, and are also usually identified by the chain where the specific CDR is located. Therefore, VHCDR3 is located in the variable domain of the antibody heavy chain where it is found, while VLCDR1 is the CDR1 from the variable domain of the antibody light chain where it is found. An antibody that binds to LHR will have a specific VH region sequence and a VL region sequence, and therefore has a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions in CDRs are directly involved in antigen binding. These positions within the CDRs are referred to as specificity determining residues (SDRs).

"Framework regions" (hereinafter referred to as FR) are those variable domain residues that are different from the CDR residues. Each variable domain generally has four FRs identified as FR1, FR2, FR3, and FR4. If CDR is defined according to Kabat, the light chain FR residues are approximately located at residues 1-23 (LCFR1), residues 35-49 (LCFR2), residues 57-88 (LCFR3), and residues 98-107 (LCFR4), and the heavy chain FR residues are approximately located at residues 1-30 (HCFR1), residues 36-49 (HCFR2), residues 66-94 (HCFR3), and residues 103-113 (HCFR4) in the heavy chain residues.

"Single-chain Fv" or "scFv" antibody fragments include the VH domain and VL domain of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide also includes a polypeptide linker between the VH domain and the VL domain, which allows the scFv to form a desired antigen binding structure. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" refers to a small antibody fragment with two antigen binding sites, which includes a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to pair between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and form two antigen binding sites. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

Diabodies or bispecific antibodies can be roughly divided into two categories: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules. IgG-like bsAb retains Fc-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) (Spiess et al., 2015, Mol Immunol., Vol. 67 (2): 95-106.). The Fc region of bsAb helps to purify and improve solubility and stability. IgG-like bispecific antibodies generally have a longer serum half-life because of their larger size and FcRn-mediated recycling (Kontermann et al., 2015, Bispecific antibodies. Drug Discov Today Vol. 20 (7): 838-47). The smaller size of non-IgG-like bsAbs results in enhanced tissue penetration (Kontermann et al., 2015, Bispecific antibodies. Drug Discov Today Vol. 20(7): 838-47).

According to some preferred embodiments, the bispecific antibody according to the present invention includes: (i) a first antigen binding site that is bound to AMHRII, and (ii) a second antigen binding site that is bound to a target antigen that is different from AMHRII, particularly a target antigen that can be expressed by cancer cells or immune cells (such as T cells, NK or macrophages) of a tumor environment. In some embodiments, in such bispecific antibodies, the second antigen binding site is bound to a target antigen (which is CD3) and engages T cells. The target antigen can also be PDL1 to unlock T cells or CD16 to activate NK or macrophages.

The monoclonal antibodies designated herein specifically include "chimeric" anti-AMHRII antibodies (immunoglobulins) in which a portion of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The monoclonal antibody specified herein also encompasses humanized anti-AMHRII antibodies. The non-human (for example, mouse) antibody of "humanization" form is a chimeric antibody containing the minimum sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies), wherein the residue from the recipient's hypervariable region is replaced by the residue of the hypervariable region of the non-human species (donor antibody, such as mouse, rat, rabbit or non-human primate) with desired specificity, affinity and ability. In some cases, the Fv framework region (FR) residue of human immunoglobulin is replaced by corresponding non-human residues. In addition, humanized antibodies may be included in residues that cannot be found in recipient antibodies or donor antibodies. These modifications are carried out in order to further improve antibody performance. Usually, humanized antibodies will comprise at least one, typically substantially all in two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The monoclonal anti-AMHRII antibodies specified herein also encompass anti-AMHRII human antibodies." Human antibody " is a human antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a person and/or using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art. In one embodiment, human antibodies are selected from phage libraries, wherein phage libraries express human antibodies (Vaughan et al. Nature Biotechnology 14: 309-314 (1996): Sheets et al. Proc. Natl. Acad. Sci. 95: 6157-6162 (1998)); Hoogenboom and Winter, J. MoI. Biol, 227: 381 (1991); Marks et al., J. MoI. Biol, 222: 581 (1991)). Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals (e.g., mice) in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which is very similar to that seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This method is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and in the following scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, human antibodies can be prepared by immortalization of human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes can be recovered from the individual or can be immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol, 147 (1): 86-95 (1991); and U.S. Pat. No. 5,750,373.

As used herein, "antibody mutants" or "antibody variants" refer to amino acid sequence variants of species-dependent antibodies, wherein one or more of the amino acid residues of the species-dependent antibody have been modified. Such mutants must have less than 100% sequence identity or similarity with the species-dependent antibody. In one embodiment, the antibody mutant will have an amino acid sequence with at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 95% amino acid sequence identity or similarity with the amino acid sequence of the heavy chain variable domain or light chain variable domain of the species-dependent antibody. The identity or similarity of the sequence is defined herein as the percentage of amino acid residues identical (i.e., identical residues) or similar (i.e., amino acid residues from the same group, based on common side chain properties, see below) to the species-dependent antibody residues in the candidate sequence after aligning the sequences and introducing spaces (if necessary) to achieve the maximum sequence identity percentage. N-terminal, C-terminal or internal extensions, deletions or insertions of antibody sequences outside the variable domain shall not be interpreted as affecting sequence identity or similarity.

Humanized antibodies can be produced by obtaining nucleic acid sequences encoding CDR domains and constructing humanized antibodies according to techniques known in the art. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e.g., Riechmann L. et al., 1988; Neuberger MS. et al., 1985). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting (EP 239,400; PCT Publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA et al. (1994)) and chain shuffling (U.S. Pat. No. 5,565,332). General recombinant DNA techniques for preparing such antibodies are also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

It may be desirable to modify the anti-AMHRII antibodies specified herein for effector functions, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residues can be introduced in the Fc region so that interchain disulfide bonds are formed in the region. The homodimeric antibodies thus produced can have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular toxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents, such as Wolff et al. Cancer Research 53: described in 2560-2565 (1993). Alternatively, the antibody can be engineered, it has dual Fc regions and therefore can have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989). WO00/42072 (Presta, L.) describes an antibody with improved ADCC function in the presence of human effector cells, wherein the antibody is included in an amino acid substitution in its Fc region. Preferably, the antibody with improved ADCC includes substitutions at positions 298, 333 and/or 334 (Eu numbering of residues) in the Fc region. Preferably, the Fc region of the change is a human IgG1 Fc region comprised of substitutions at one, two or three of these positions or consisting of substitutions at one, two or three of these positions. Such substitutions are optionally combined with substitutions that increase CIq binding and/or CDC.

Antibodies with altered CIq binding and/or complement dependent cytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No. 6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No. 6,528,624B1, and U.S. Pat. No. 6,538,124 (Idusogie et al.). The antibody comprises an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333, and/or 334 (Eu numbering of residues) in its Fc region.

In some embodiments, the AMHRII binding agent encompasses a glycoengineered anti-AMHRII antibody.

As used herein, the term "glycoengineering" refers to any art-recognized method for altering the glycoform profile of a binding protein composition. Such methods include expressing the binding protein composition in a genetically engineered host cell (e.g., CHO cell) that is genetically engineered to express a heterologous glycosyltransferase or glycosidase. In other embodiments, the glycoengineering method includes culturing the host cell under conditions that favor a specific glycoform profile.

As used herein, "glycoengineered antibodies" encompass (i) antibodies comprising a high galactosylation Fc fragment, (ii) antibodies comprising a low mannosylation Fc fragment (which encompasses a mannosylated Fc fragment), and (iii) antibodies comprising a low fucosylation Fc fragment (which encompasses a fucosylated Fc fragment). As used herein, glycoengineered fragments encompass Fc fragments with altered glycosylation selected from the group comprising one or more of the following altered glycosylations: (i) high galactosylation, (ii) low mannosylation, and (iii) low fucosylation. As a result, the glycoengineered Fc fragments from the anti-AMHRII antibodies used according to the present invention encompass illustrative examples of high galactosylation Fc fragments, low mannosylation Fc fragments, and low fucosylation Fc fragments.

Those skilled in the art can refer to known techniques for obtaining anti-AMHRII antibodies comprising a hypergalactosylated Fc fragment, a hypomannosylated Fc fragment, and a hypofucosylated Fc fragment known to bind to an Fc receptor with higher affinity than a non-modified Fc fragment.

Glycoengineered anti-AMHRII antibodies encompass anti-AMHRII antibodies comprising a hypofucosylated Fc fragment (also referred to as a "low-fucose" Fc fragment).

Immunoconjugates, especially antibody-drug conjugates (ADCs)

AMHRII binding agents that can be used for the purposes of the present invention encompass antibodies specified herein conjugated to cytotoxic agents such as chemotherapeutic agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof) or radioactive isotopes (i.e., radioconjugates). Such antibody conjugates encompass those described in PCT Application No. WO 2017/025458. PCT Application No. WO 2017/025458 discloses, inter alia, anti-AMHRII 3C23K antibodies, as well as 3C23K ADC conjugates, which herein show in vivo anticancer activity against non-gynecological human cancers.

Cytotoxic agents encompass enzymatically active toxins. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modisin A chain, alpha-sarcin, Aleurites fordii proteins, dianthus proteins, pokeweed proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitocin, restrictocin, phenomycin, enomycin and trichothecenes.

A variety of radionuclides are available for the production of radioconjugate antibodies.

Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents such as those disclosed in PCT Application No. WO2017/025458.

Preferred immunoconjugates of anti-AMHRII ADC antibody conjugates are those described in PCT Application No. WO 2017/025458.

CAR cells, including CAR T cells, CAR NK cells, and CAR macrophages

In some embodiments, the human AMHRII binding agent is an AMHRII binding receptor or an AMHRII binding receptor expressing cell, particularly an AMHRII binding receptor expressing CAR T cell, an AMHRII binding receptor NK cell or an AMHRII binding receptor expressing CAR macrophage.

Therefore, in some embodiments, the human AMHRII binding agent is an AMHRII binding engineered receptor, most preferably an AMHRII binding engineered receptor whose AMHRII binding region is derived from the monoclonal anti-AMHRII antibody disclosed in the present specification.

Typically, AMHRII binding engineered receptors are composed of chimeric antigen receptors (CARs) including the following: (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain, and wherein the extracellular domain is derived from the AMHRII binding portion of the anti-AMHRII monoclonal antibody disclosed in this specification. In some embodiments, the extracellular domain of the AMHRII binding engineered receptor comprises (i) an antibody VH chain comprising a CDR derived from an anti-AMHRII monoclonal antibody disclosed herein and (ii) an antibody VL chain comprising a CDR derived from an anti-AMHRII monoclonal antibody disclosed herein. In some embodiments, the extracellular domain of the AMHRII binding engineered receptor comprises a VH chain and a VL chain of an anti-AMHRII monoclonal antibody disclosed herein. In some embodiments, the extracellular domain of the AMHRII binding engineered receptor is a ScFv comprising CDRs derived from the VH chain and the CH chain of the anti-AMHRII monoclonal antibody disclosed in this specification, respectively. In some embodiments, the AMHRII-binding engineered receptor extracellular domain is a ScFv comprising a VH chain and a CH chain respectively derived from an anti-AMHRII monoclonal antibody disclosed in the present specification.

Also contemplated herein are AMHRII binding agents composed of cells expressing such AMHRII binding receptors, particularly CAR T cells, CAR NK cells or CAR macrophages expressing such AMHRII binding receptors.

As used herein, the term "chimeric antigen receptor" (CAR) refers to a fusion protein including an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes also referred to as a "chimeric receptor", "T body" or "chimeric immune receptor (CIR)". "Capable of binding to the extracellular domain of AMHRII" means any oligopeptide or polypeptide that can bind to AMHRII. "Intracellular domain" means any oligopeptide or polypeptide known in cells to be used as a domain for transmitting signals to cause activation or inhibition of biological processes. "Transmembrane domain" means any oligopeptide or polypeptide known to cross cell membranes and can be used to connect an extracellular domain and a signaling domain. The chimeric antigen receptor may optionally include a "hinge domain" used as a linker between the extracellular domain and the transmembrane domain.

CAR T cells are genetically engineered autologous T cells in which a single-chain antibody fragment (scFv) or a ligand is attached to a T cell signaling domain that can promote T cell activation (Maher, J. (2012) ISRN Oncol. 2012:278093; Curran, K.J. et al. (2012) J. Gene Med. 14:405-415; Fedorov, V.D. et al. (2014) Cancer J. 20:160-165; Barrett, D.M. et al. (2014) Annu. Rev. Med. 65:333-347).

"Intracellular signaling domain" means the part of CAR that is found inside T cells or engineered to be found. "Intracellular signaling domain" may or may not also include a "transmembrane domain" that anchors CAR in the plasma membrane of T cells. In one embodiment, the "transmembrane domain" and the "intracellular signaling domain" are derived from the same protein (e.g., CD3ζ); in other embodiments, the intracellular signaling domain and the transmembrane domain are derived from different proteins (e.g., the transmembrane domain of CD3ζ and the intracellular signaling domain of the CD28 molecule, and vice versa).

"Costimulatory endodomain" means an intracellular signaling domain or a fragment thereof derived from a T cell co-stimulatory molecule. A non-limiting list of T cell co-stimulatory molecules includes CD3, CD28, OX-40, 4-1BB, CD27, CD270, CD30, and ICOS. A co-stimulatory endodomain may or may not include a transmembrane domain from the same or different co-stimulatory endodomain.

“Extracellular antigen binding domain” means the portion of CAR that specifically recognizes AMHRII and binds to AMHRII.

In a preferred embodiment, the "extracellular binding domain" is derived from an anti-AMHRII monoclonal antibody. For example, the "extracellular binding domain" may include all or part of the Fab domain from a monoclonal antibody. In certain embodiments, the "extracellular binding domain" includes the complementary determining region of a specific anti-AMHRII monoclonal antibody. In another embodiment, the "extracellular binding domain" is a single-chain variable fragment (scFv) obtained from an anti-AMHRII monoclonal antibody specified herein.

In a preferred embodiment, the extracellular binding domain is derived from any one of the anti-AMHRII monoclonal antibodies described in the present specification, particularly from the 3C23K anti-AMHRII monoclonal antibody.

I. Extracellular antigen binding domain

In one embodiment, the CAR of the invention comprises an extracellular antigen binding domain from one of the anti-AMHRII monoclonal antibodies described herein.

In one embodiment, the extracellular binding domain comprises the following CDR sequences:

-CDRL-1: RASX1X2VX3X4X5A (SEQ ID NO. 65), wherein X1 and X2 are independently S or P, X3 is R or W or G, X4 is T or D, and X5 is I or T;

-CDRL-2 is PTSSLX6S (SEQ ID NO. 66), wherein X6 is K or E; and

-CDRL-3 is LQWSSYPWT (SEQ ID NO. 67);

-CDRH-1 is KASGYX7FTX8X9HIH (SEQ ID NO. 68), wherein X7 is S or T, X8 is S or G, and X9 is Y or N;

-CDRH-2 is WIYPX10DDSTKYSQKFQG (SEQ ID NO. 69), wherein X10 is G or E, and

-CDRH-3 is GDRFAY (SEQ ID NO. 70).

II. Linker between the VL and VH domains of κMab scFv

In another embodiment, the anti-AMHRII VL is connected to the anti-AMHRII VH via a flexible linker. Specifically, the flexible linker is a glycine/serine linker of about 10 to about 30 amino acids (e.g., 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids) and comprises the structure (Gly4Ser) ³ .

III. Spacer between the extracellular antigen binding domain and the intracellular signaling domain

The extracellular antigen binding domain is connected to the intracellular signaling domain by using a "spacer". The spacer is designed to be flexible enough to allow the antigen binding domain to be oriented in a manner that facilitates antigen recognition and binding. The spacer can be derived from the anti-AMHRII immunoglobulin itself and can include an IgG1 hinge region or a CH2 region and/or a CH3 region of an IgG.

IV. Intracellular signaling domain

The intracellular signaling domain contains all or part of the CD3 chain. CD3, also known as CD247, together with the CD4 T cell co-receptor or CD8 T cell co-receptor, is responsible for coupling extracellular antigen recognition to the intracellular signaling cascade.

In addition to including the CD3ζ signaling domain, the inclusion of co-stimulatory molecules has been shown to enhance CAR T cell activity in murine models and clinical trials. Several have been studied, including CD28, 4-IBB, ICOS, CD27, CD270, CD30, and OX-40.

In certain embodiments, a method for producing CAR-expressing cells is disclosed, the method comprising the following or alternatively consisting essentially of the following: (i) transducing a separated cell population with a nucleic acid sequence encoding CAR, and (ii) selecting a cell subpopulation successfully transduced with the nucleic acid sequence of step (i). In some embodiments, the separated cells are T cells, animal T cells, mammalian T cells, feline T cells, canine T cells or human T cells, thereby producing CAR T cells. In certain embodiments, the separated cells are NK cells, for example, animal NK cells, mammalian NK cells, feline NK cells, canine NK cells or human NK cells, thereby producing CAR NK cells.

Therapeutic applications of CAR T cells, CAR NK T cells, and CAR macrophages

CAR cells including CAR T cells, CAR NK cells and CAR macrophages described herein can be used to treat lung tumors expressing AMHRII. CAR cells of the present invention are preferably used to treat lung tumors expressing AMHRII in patients with lung cancer described herein, especially non-small cell lung cancer or small cell lung cancer.

The CAR cells of the present invention can be administered alone or in combination with diluents, known anti-cancer therapeutics and/or other components (such as cytokines) or other cell populations with immunostimulatory properties.

The method aspects of the invention relate to methods for inhibiting tumor growth in a subject in need thereof and/or for treating a cancer patient in need thereof. In some embodiments, the tumor is a solid lung tumor.

CAR cells as disclosed herein can be administered alone or in combination with diluents, known anticancer therapeutic agents and/or other components (such as cytokines) or other cell populations with immunostimulation. They can be first-line therapy, second-line therapy, third-line therapy, fourth-line therapy or another therapy. They can be combined with other therapies. Such non-limiting examples include chemotherapy or biologics. The appropriate treatment regimen will be determined by the attending physician or veterinarian.

The pharmaceutical composition comprising the CAR of the present invention can be administered in a manner suitable for the disease to be treated or prevented. Although the appropriate dosage can be determined by clinical trials, the amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease.

Therapeutic applications

As disclosed elsewhere in this specification, the AMHRII binders disclosed herein cover (i) the anti-AMHRII antibodies disclosed herein, (ii) the antibody drug conjugates disclosed herein and (iii) the CAR cells disclosed herein (including CAR T cells, CAR NK cells and CAR macrophages), and the AMHRII binders are composed of active ingredients that can be used to prevent or treat lung cancer expressing AMHRII, in particular non-small cell lung cancer (NSCLC), more precisely NSCLC selected from the group including: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC and neuroendocrine NSCLC.

Cancer treatment methods using anti-tumor antigen antibodies or anti-tumor antigen CAR cells are well known to those skilled in the art.

In some embodiments, cancer patients are tested to determine whether their tumor cells express AMHRII at their surface prior to treatment with an AMHRII binding agent (such as an anti-AMHRII antibody, anti-AMHRII ADC, or anti-AMHRII CAR T cells).

Such preliminary tests for detecting membrane expression of AMHRII are preferably used for treating lung cancers that express AMHRII at low frequencies. In contrast, such preliminary tests for detecting membrane expression of AMHRII may not be performed for treating cancers that express AMHRII at high frequencies (such as epidermoid NSCLC as an illustration).

Therefore, in some embodiments, the present invention relates to the use of an AMHRII binding agent as specified herein for preventing or treating an individual with AMHRII-positive lung cancer, including non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC.

The present invention relates to the use of an AMHRII binder for the preparation of a medicament for preventing or treating an individual suffering from AMHRII-positive lung cancer, including lung cancer, including non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group consisting of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCL, pleomorphic cell carcinoma NSCLC C and neuroendocrine NSCLC.

The present invention also relates to a method for preventing or treating an individual suffering from AMHRII-positive lung cancer, including non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group consisting of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, pleomorphic cell carcinoma NSCLC and neuroendocrine NSCLC, wherein the method comprises the step of administering an anti-AMHRII binder to the individual.

By detecting the method for the cell surface AMHRII protein expression on the lung cancer tissue sample previously obtained from the individual, the individual can be designated as the individual suffering from the positive cancer of AMHRII. The detection of cell surface AMHRII protein expression can be carried out according to multiple methods well known to those skilled in the art. The cell surface AMHRII protein expression detection method particularly encompasses immunohistochemical methods and fluorescence activated cell sorting methods, as shown in the examples herein.

The present invention also relates to a method for determining whether an individual is suitable for lung cancer treatment with an AMHRII binder (i.e., whether the individual responds to lung cancer treatment with an AMHRII binder), wherein the method includes the step of determining whether a lung tumor tissue sample previously obtained from the individual expresses AMHRII protein at the cell surface.

Therefore, the present invention also relates to a method for determining whether an individual suffering from lung cancer, in particular non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC and neuroendocrine NSCLC is suitable for cancer treatment with an AMHRII binding agent (i.e. responds to cancer treatment with an AMHRII binding agent), wherein the method comprises the following steps:

a) determining whether cancer cells from said patient express AMHRII at their membrane, and

b) inferring that if membrane expression of AMHRII by the lung cancer cells is determined in step a), the patient is suitable for (ie responds to) lung cancer treatment with an AMHRII binding agent.

In a preferred embodiment of the method, in step b), when (i) in step a) the AMHRII expression score value is determined and when (ii) the AMHRII expression score value is a threshold score value or higher, it is inferred that the patient is suitable for (i.e., responsive to) lung cancer treatment with an AMHRII binding agent. The AMHRII score value is most preferably calculated by using formula (I) described elsewhere in this specification.

Therefore, according to a preferred embodiment, step a) of the method is performed by an immunohistochemical method, such as shown in the examples herein.

The cancer cells used in step a) are usually derived from a biopsy tissue sample previously collected from said cancer patient.

Preferably, step a) is performed by using an anti-AMHRII antibody selected from those specifically described in the present specification, in particular the 3C23K antibody, the AMHRII binding of which can be detected according to well-known antibody detection techniques, such as those disclosed in the examples herein, by using a labeled secondary antibody.

Preferably, when the scoring method allowing determination of the E score value is performed according to the following formula (I), when a membrane AMHRII expression score value of 1.0 or greater is determined in a cancer cell sample derived from a cancer patient suffering from a lung cancer included in the above-listed lung cancer group, the patient is determined to be suitable for (i.e., responsive to) lung cancer treatment using an AMHRII binding agent:

E score = frequency x AMHRII_level, where

-E score means the membrane AMHRII expression score value of a given cancer cell sample,

- frequency means the frequency of cells contained in said lung cancer cell sample in which membrane AMHRII expression is detected, and

-AMHRII_level means the level of AMHRII membrane expression of AMHRII expressing cells contained in said given lung cancer cell sample.

Therefore, the present invention also relates to a method for treating a patient suffering from non-small cell lung cancer (NSCLC), wherein the method comprises the following steps:

a) determining whether a tumor tissue sample previously obtained from the individual expresses AMHRII protein at the cell surface, and

b) if cell surface expression of AMHRII has been determined in step a), treating the individual with an AMHRII binding agent.

In a most preferred embodiment, in step a), AMHRII expression is determined when the tumor sample has a membrane AMHRII expression score value "E score" of 1.0 or greater (including E score values of 1.5 or greater) calculated according to formula (I) above.

In the most preferred embodiment of the above method, the AMHRII binding agent is composed of an anti-AMHRII antibody or a fragment thereof as specified herein, or is composed of a CAR cell (eg, a CAR T cell or a CAR NK cell) as specified herein.

In some embodiments, the AMHRII binding agent is used as the sole anti-cancer active ingredient.

In some other embodiments, the anti-cancer treatment with said AMHRII binding agent further comprises subjecting said individual to one or more additional anti-cancer therapies including radiation therapy treatment and chemotherapeutic agent treatment.

Thus, according to such other embodiments, anti-cancer treatment with the AMHRII binding agent further comprises administering to the individual one or more additional anti-cancer active ingredients.

Combination therapy

As shown in the examples herein, effective anti-lung cancer therapies encompass those in which anti-AMHRII monoclonal antibodies are combined with one or more different anticancer agents. The examples herein illustrate combination therapies for lung cancer in which anti-AMHRII antibodies are combined with docetaxel or with a combination of cisplatin and gemcitabine.

"Anticancer" is defined as any molecule that can interfere with the biosynthesis of macromolecules (DNA, RNA, proteins, etc.) or inhibit cell proliferation or cause cell death (e.g., by apoptosis or cytotoxicity). Among anticancer agents, mention may be made of alkylating agents, topoisomerase inhibitors and intercalators, antimetabolites, cleaving agents, agents that interfere with tubulin, monoclonal antibodies.

"Pharmaceutically acceptable vehicle" refers to a nontoxic substance that is compatible with biological systems such as cells, cell cultures, tissues or organisms.

According to a particular aspect, the present invention relates to a pharmaceutical composition comprising as active ingredients an anti-cancer agent and an antibody binding to AMHR-II, in particular an anti-AMHRII antibody as described herein, in combination with a pharmaceutically acceptable vehicle.

In some embodiments, the present invention relates to a pharmaceutical composition comprising, as active ingredients, an anti-cancer agent and an antibody that binds to AMHR-II, particularly an anti-AMHRII antibody described herein, in combination with a pharmaceutically acceptable vehicle.

In some embodiments, the present invention relates to a pharmaceutical composition comprising an anticancer agent as an active ingredient and an antibody that binds to AMHR-II in combination with a pharmaceutically acceptable vehicle, wherein the anticancer agent is selected from the group consisting of docetaxel, cisplatin, gemcitabine, and a combination of cisplatin and gemcitabine.

Other anticancer agents that can be used in combination with anti-AMHRII antibodies include paclitaxel or platinum salts (such as oxaliplatin, cisplatin and carboplatin).

The anticancer agent may also be selected from chemotherapeutic agents other than platinum salts, small molecules, monoclonal antibodies or other antiangiogenic peptibodies.

Chemotherapeutic agents other than platinum salts include intercalating agents (blocking DNA replication and transcription) such as anthracyclines (doxorubicin, pegylated liposomal doxorubicin), topoisomerase inhibitors (camptothecin and derivatives: karenitecin, topotecan, irinotecan), or SJG-136, inhibitors of histone deacetylases (vorinostat, belinostat, valproic acid), alkylating agents (bendamustine, glufosfamide, temozolomide), antimitotic plant alkaloids such as taxanes (docetaxel, paclitaxel), vinca alkaloids (vinorelbine), epothilones (ZK-epothilone, ixabepilone), antimetabolites (gemcitabine, elcitabine, capecitabine), kinesin (KSP) inhibitors (ispins), trabectedin or orebrine (comprestatin A-4 derivative).

Among the small molecules there are poly (ADP-ribose) polymerase (PARP) inhibitors: olaparib, eniparib, veliparib, rucaparib, CEP-9722, MK-4827, BMN-673, kinase inhibitors such as tyrosine kinase inhibitors (TKI), among which there may be mentioned anti-VEGFR molecules (sorafenib, sunitinib, cediranib, vandetanib, pazopanib, BIBF1120, semacanib, cabozantinib, motesanib), anti-HER2/EGFR molecules (erlotinib, gefitinib, lapatinib), anti-PDGFR molecules (imatinib, BIBF 1120), anti-FGFR molecules (BIBF 1120), Aurora kinase/tyrosine kinase inhibitors (ENMD-2076), Src/Abl kinase inhibitors (saracatinib), or also perifosine, terolimus (mTOR inhibitor), avosidib (cyclin-dependent kinase inhibitor), voraserti (inhibitor of PLK1 (Paul-like kinase 1) protein), LY2606368 (inhibitor of checkpoint kinase 1 (chk 1)), GDC-0449 (Hedgehog pathway inhibitor), zilpotentan (antagonist of ETA-receptor), bortezomib, carfilzomib (proteasome inhibitor), cytokines (such as IL-12, IL-18, IL-21, INF-α, INF-γ).

Among the antibodies, mention may be made of anti-VEGF: bevacizumab, anti-VEGFR: ramucirumab, anti-HER2/EGFR: trastuzumab, pertuzumab, cetuximab, panitumumab, MGAH22, matuzumab, anti-PDGFRα: IMC-3G3, anti-folate receptor: faratumumab, anti-CD27: CDX-1127, anti-CD56: BB-10901, anti-CD105: TRC105, anti-CD276: MGA271, anti-AGS-8: AGS-8M4, anti-DRS: TRA-8, anti-HB-EGF: KHK2866, anti-mesothelin: amatuximab, BAY 94-9343 (immunotoxin), catumaxomab (EpCAM/CD3 bispecific antibody), anti-IL2R: daclizumab, anti-IGF-1R: ganizumab, anti-CTLA-4: ipilimumab, anti-PD1: nivolumab and pembrolizumab, anti-CD47: Weissman B6H12 and Hu5F9, Novimmune 5A3M3, INHIBRX 2A1, Frazier VxP037-01LC1 antibody, anti-Lewis Y: Hu3S193, SGN-15 (immunotoxin), anti-CAl25: ogavuzumab, anti-HGF: relottolumab, anti-IL6: siltuximab, anti-TR2: tegtuzumab, anti-α5β1 integrin: volotuximab, anti-HB-EGF: KHK2866. The anti-angiogenic peptide antibody is selected from AMG 386 and CVX-241.

More specifically, described herein is a pharmaceutical composition comprising an anticancer agent as an active ingredient and an antibody that binds to AMHR-II in combination with a pharmaceutically acceptable vehicle, wherein the anticancer agent is selected from the group consisting of docetaxel, cisplatin, gemcitabine, and a combination of cisplatin and gemcitabine.

Even more specifically, described herein are pharmaceutical compositions comprising as active ingredients an anticancer agent and an antibody that binds to AMHR-II in combination with a pharmaceutically acceptable vehicle, wherein the mutated humanized monoclonal antibody referred to herein as 3C23K and the anticancer agent are selected from the group consisting of docetaxel, cisplatin, gemcitabine, and a combination of cisplatin and gemcitabine.

In particular aspects, described herein are pharmaceutical compositions comprising as active ingredients an anticancer agent and an antibody that binds AMHR-II in combination with a pharmaceutically acceptable vehicle in a formulation intended for administration by the intravenous or intraperitoneal route.

In another specific aspect, the present invention relates to the use of a composition comprising an anticancer agent and an antibody binding to AMHR-II as a pharmaceutical product in a formulation intended for administration by the intravenous or intraperitoneal route in the prevention or treatment of lung cancer.

In another specific aspect, the present invention relates to the use of a composition as a pharmaceutical product in the prevention or treatment of lung cancer, the composition comprising an anticancer agent and an antibody binding to AMHR-II, the monoclonal antibody and the anticancer agent being intended for separate, simultaneous or sequential administration.

The antibody and anticancer agent may be combined in the same pharmaceutical composition, or may be used in the form of separate pharmaceutical compositions for simultaneous or sequential administration. In particular, the products may be administered separately, ie simultaneously or independently, for example with a time interval.

More specifically, the present invention relates to the use of a composition as a pharmaceutical product in the prevention or treatment of lung cancer, the composition comprising an anticancer agent and an antibody that binds to AMHR-II, wherein the antibody and the anticancer agent are combined in the same pharmaceutical composition.

According to another specific aspect, the present invention relates to the use of a composition as a pharmaceutical product in the prevention or treatment of lung cancer, the composition comprising an anti-cancer agent and an antibody that binds to AMHR-II, wherein the therapeutically effective amount of the anti-AMHRII antibody administered to the patient ranges from about 0.07 mg to about 35,000 mg, preferably from about 0.7 mg to about 7,000 mg, preferably from about 0.7 mg to about 1,400 mg, preferably from about 0.7 mg to about 700 mg and more preferably from about 0.7 mg to about 70 mg.

According to another specific aspect, the present invention relates to the use of a composition as a pharmaceutical product in the prevention or treatment of lung cancer, the composition comprising an anticancer agent and an antibody that binds to AMHR-II, wherein the therapeutically effective amount of the anticancer agent administered to the patient ranges from about 10 mg to about 700 mg, preferably ranges from about 20 mg to about 350 mg and preferably about 110 mg.

According to another specific aspect, the present invention relates to the use of a composition as a pharmaceutical product in the prevention or treatment of lung cancer, wherein the composition comprises an anticancer agent and an antibody that binds to AMHR-II, wherein the therapeutically effective amount of the antibody administered to the patient is about 70 mg and the dose of the anticancer agent administered to the patient is about 110 mg.

In a preferred embodiment, the dosage of the anticancer agent (particularly docetaxel or a combination of cisplatin and gemcitabine) ranges from about 0.01 mg/kg to about 500 mg/kg per day, such as 0.1 mg/kg to 300 mg/kg, or about 0.1 mg to 20 g.

As a variant, it is also possible to administer a higher initial loading dose, followed by one or more lower doses. In another variant, it is also possible to administer a less high initial loading dose, followed by one or more higher doses.

In specific embodiments, the anti-AMHRII antibody and the anticancer agent may be used in an antibody/anticancer agent weight ratio ranging from about 10/1 to about 0.01/1, particularly about 10/1 to about 0.05/1, or about 5/1 to about 0.1/1.

Illustratively, anti-AMHRII antibody and docetaxel can be used in a 1/1 antibody/docetaxel weight ratio, as shown in the examples herein.

Still exemplarily, anti-AMHRII antibody and cisplatin can be used in an antibody/cisplatin weight ratio of 4/1, as shown in the examples herein.

Still exemplarily, anti-AMHRII antibody and gemcitabine may be used at an antibody/gemcitabine weight ratio of 0.2/1, as shown in the examples herein.

The invention also describes a product comprising an antibody binding to human anti-Mullerian hormone type II receptor (AMHR-II) and an anticancer agent in the form of a combined preparation for simultaneous, sequential or separate use as a pharmaceutical product intended for the prevention or treatment of lung cancer expressing AMHRII.

[00136] AMHRII binding agents as disclosed herein, particularly anti-AMHRII antibodies disclosed herein, can be administered in a variety of ways, including oral administration, subcutaneous administration, and intravenous administration.

The term "therapeutically effective amount" refers to the amount of a drug that effectively treats a disease or condition in a mammal. In the case of cancer, the therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of a tumor; inhibit (i.e., slow down, preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and/or alleviate one or more symptoms related to the condition to a certain extent. To a certain extent, the drug can prevent growth and/or kill existing cancer cells, it can inhibit cell growth and/or have cytotoxicity. For cancer therapy, in vivo efficacy can be measured by, for example, assessing the duration of survival, the duration of progression-free survival (PFS), the duration of response rate (RR), the duration of response, and/or the quality of life.

Therapeutic formulations of agents (e.g., antibodies) used according to the invention are prepared in the form of lyophilized formulations or aqueous solutions for storage by mixing the antibodies having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 0 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG).

The active ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

Preparations for in vivo administration may be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Pharmaceutical compositions as described herein can be administered by any suitable route of administration, for example, by parenteral, oral, sublingual, vaginal, rectal or transdermal routes, preferably by intravenous, subcutaneous or intradermal injection. Intramuscular, intraperitoneal, intrasynovial, intrathecal or intratumoral injection is also possible. Injection can be performed in the form of a bolus or by continuous infusion. When the antibody composition and the anticancer agent composition are administered separately, these compositions can be the same or different forms of administration.

Preparations for parenteral administration can include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) or injectable organic esters (such as ethyl oleate). Aqueous vehicles include water, alcohol/aqueous solutions and emulsions or suspensions.

The pharmaceutical composition as described herein advantageously comprises one or more pharmaceutically acceptable excipients or vehicles. For example, saline, physiological solutions, isotonic solutions, buffer solutions, etc., which are compatible with pharmaceutical use and known to those skilled in the art, can be mentioned. The composition may contain one or more reagents or vehicles selected from dispersants, solubilizers, stabilizers, preservatives, etc. The reagents or vehicles that can be used in the formulation (liquid and/or injectable and/or solid) are particularly methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, gum arabic, etc. The composition can be formulated in the form of injectable suspensions, gels, oils, tablets, suppositories, powders, hard gelatin capsules, soft capsules, etc.

According to a specific aspect, the present invention relates to a pharmaceutical composition comprising as active ingredients an anti-cancer agent and an anti-AMHRII antibody in combination with a pharmaceutically acceptable vehicle, wherein the therapeutically effective amount of the antibody administered to the patient ranges from about 0.07 mg to about 35000 mg, preferably from about 0.7 mg to about 7000 mg, preferably from about 0.7 mg to about 1400 mg, preferably from about 0.7 mg to about 700 mg and more preferably from about 0.7 mg to about 70 mg.

The dosage of the active ingredient depends particularly on the method of administration and is easily determined by those skilled in the art. In one or more weekly administrations for several weeks or months, the therapeutically effective amount (unit dose) of the antibody may vary from 0.01 mg/kg to 500 mg/kg, preferably from 0.1 mg/kg to 500 mg/kg, preferably from 0.1 mg/kg to 100 mg/kg, preferably from 0.1 mg/kg to 20 mg/kg, preferably from 0.1 mg/kg to 10 mg/kg and more preferably from 1 mg/kg to 10 mg/kg. Therefore, the effective unit dose can be easily derived from the dose calculated for an "average" patient weighing 70 kg.

According to another specific aspect, the present invention relates to a pharmaceutical composition comprising as active ingredients an anti-cancer agent and an anti-AMHRII antibody in combination with a pharmaceutically acceptable vehicle, wherein the therapeutically effective amount of the anti-cancer agent administered to the patient is in the range of about 10 mg to about 700 mg, preferably in the range of about 20 mg to about 350 mg, and preferably about 110 mg.

The dosage of the anticancer agent depends inter alia on the method of administration and is readily determined by one skilled in the art. In one or more weekly administrations over a period of weeks or months, the therapeutically effective amount (unit dose) may vary from 0.2 mg/m ² to 10 g/m ² , preferably from 0.2 mg/m ² to 1 g/m ² , preferably from 2 mg/m ² to 1 g/m ² , preferably from 20 mg/m ² to 1 g/m ² and more preferably from 20 mg/m ² to 0.5 g/m ^2. Thus, the effective unit dose may be derived from the dose calculated for an "average" patient having a body surface area of approximately 1.8 m ² .

According to an even more specific aspect, the present invention is directed to a pharmaceutical composition comprising as active ingredients an anti-cancer agent and an anti-AMHRII antibody in combination with a pharmaceutically acceptable vehicle, wherein the therapeutically effective amount of the anti-cancer agent administered to the patient is about 110 mg and the therapeutically effective amount of the antibody administered to the patient is about 70 mg.

The present invention also describes the use of a composition as a pharmaceutical product in the prevention or treatment of lung cancer expressing AMHRII, the composition comprising an anticancer agent and an anti-AMHRII antibody that binds to human anti-Mullerian hormone type II receptor (AMHR-II).

The present invention is further illustrated by, but not limited in any way to, the following examples.

Example

Example 1: Differential AMHRII gene expression and AMHRII protein expression

A. Materials and Methods

A.1. Cell lines and cultures

COV434 WT cell line (ECACC No. 07071909) was maintained in DMEM/GlutaMax (Gibco) supplemented with 10% FBS, penicillin 100 U/ml and streptomycin 100 μg/ml. Geneticin (Gibco) was added at 400 μg/ml for COV434 MISRII transfected cell lines. Erythroleukemia K562 cell line ( CCL-243 ^™ ) were cultured in suspension in IMDM medium (Sigma-Aldrich) supplemented with 10% FBS and penicillin/streptomycin and maintained at a density of 1 x 10 ⁵ to 1 x 10 ⁶ cells/ml in T75 flasks. CRL-11732 ^™ , ovarian serous adenocarcinoma) were cultured in a 1:1 mixture of MCDB 105 medium (Sigma-Aldrich) containing a final concentration of 1.5 g/l sodium bicarbonate and medium 199 (Sigma-Aldrich) containing a final concentration of 2.2 g/l sodium bicarbonate (supplemented with 15% FBS and penicillin/streptomycin). NCI-H295R cell line (adrenocortical carcinoma, CRL-2128 ^™ ) were maintained in DMEM:F12 medium (Sigma-Aldrich) supplemented with iTS ⁺ Premix (Corning), 2.5% Nu-Serum (Falcon) and penicillin/streptomycin. Cells were grown at 37°C in a humidified atmosphere with 8% _CO2 , and the medium was changed once or twice a week depending on the cell line.

A.2. Relative quantification of AMHR2 mRNA by RT-qPCR

Extract RNA using the manufacturer's instructions. Total RNA from 1x10 ⁶ to 5x10 ⁶ cell pellets was prepared using the Plus RNA purification kit (Ambion). Briefly, after phenol/chloroform extraction, RNA from lysed cells was adsorbed on a silica matrix, treated with DNase, and then washed and eluted with 30 μl of RNase-free water. RNA concentration and quality were assessed using a spectrophotometer (NanoDrop, ThermoFisher Scientific).

cDNA synthesis. RNA (1 μg) was reverse transcribed using the Maxima H Minus First Strand cDNA Synthesis Kit (Ambion) and an oligo-dT primer by incubating at 25°C for 10 min for priming and 50°C for 15 min for reverse transcription, followed by incubation at 85°C for 5 min for reverse transcriptase inactivation.

Quantitative PCR. Quantitative PCR was performed in a Light Cycler 480 (Roche) in a 96-well microplate using Luminaris Color HiGreen qPCR Master Mix (Ambion) in a final volume of 20 μl. The following primers were used: for AMHR2, forward 5'-TCTGGATGGCACTGGTGCTG-3' (SEQ ID NO. 71) and reverse 5'-AGCAGGGCCAAGATGATGCT-3' (SEQ ID NO. 72); for TBP, forward 5'-TGCACAGGAGCCAAGAGTGAA-3' (SEQ ID NO. 73) and reverse 5'-CACATCACAGCTCCCCACCA-3' (SEQ ID NO. 74). Amplification was performed using cDNA template (100 ng equivalent RNA) and the following protocol: UDG pretreatment at 50 ° C for 2 min, denaturation at 95 ° C for 10 min, and then 40 cycles of 15 s at 95 ° C/30 s at 60 ° C/30 s at 70 ° C. Melting curve analysis was performed at the end of each experiment to control the absence of genomic DNA and dimer primers. Each cDNA sample and control ("no template sample" and "no reverse transcript RNA") were tested in duplicate. The mean value of the cycle threshold (Ct) was calculated, and the relative quantification (RQ) of AMHR2 was expressed as 2 ^{- △ △ Ct} , where △ △ Ct = △ Ct _sample - △ Ct _calibration and △ Ct = Ct _AMHR2 - Ct _TBP . HCT116 samples were used as calibration products, and TBP was used as a housekeeping gene for normalization.

Table 2 below depicts the AMHRII expression levels in the cell lines tested using the Q-PCR method described above.

Table 2

A.3. Evaluation of membrane AMHR2 expression by flow cytometric analysis

For fluorescence activated cell sorting (FACS) analysis, 4 x 10 ⁵ cells were incubated with 25 μg/ml of 3C23K at 4°C for 30 min. After washing with PBS-BSA2%, the primary antibody was detected by an anti-species secondary antibody conjugated to a fluorophore. 3C23K was detected by anti-human F(ab') ₂ conjugated to phycoerythrin (1:1000, Beckman-Coulter, IM0550). After washing with PBS, FACS analysis of resuspended cells was achieved in the FL2 channel of a BD Accuri ^TM C6 flow cytometer (BD Bioscience).

B. Results

The results are depicted in Figure 2. The results show that although the COV434-WT cell line has a significant membrane expression level of human AMHRII protein, the recombinant cell line COV434-WT (AMHRII gene expression level measured for the cell line NCI-H295R is approximately 3%).

These results indicate that there is absolutely no correlation between AMHRII gene expression and membrane AMHRII protein expression.

Example 2: AMHRII expression in lung cancer (human tumor samples)

A. Materials and Methods

A.1. Objectives

For immunohistochemical studies of human cancer cell xenografts (PDX) in mice using biotinylated 3C23K monoclonal antibody to detect anti-Mullerian hormone type 2 receptor (AMHR2) expression.

A.2. Plan and Method

- Cell lines: fixed in formaldehyde-acetate alcohol (AFA) with cell block configuration.

- Human tumors: External samples were fixed in formalin and slides from Institut Curie were fixed in AFA.

- Immunohistochemistry (IHC) techniques are possible after deparaffinization of the samples and exposure at pH 9 (microwave EZ Retriever 15' at 90°C followed by cooling during 20').

- Anti-Mullerian hormone type II receptor detection revealed by immunoperoxidase technique and DAB chromogenic substrate.

- After blocking endogenous peroxidase activity, slides were incubated with diluted biotinylated primary antibodies (1/800, 8 μg/mL) at room temperature for 90 minutes. Tissue sections were then washed with PBS and incubated with avidin/biotin ABC [carrier] complex for 30 minutes. Immunoreactive signals were detected using DAB substrate solution (DAB + substrate buffer / liquid DAB + chromogen, 10 minutes incubation). Finally, sections were lightly counterstained with Mayer's hematoxylin (Lilly's modified).

- Negative controls were obtained by replacing the primary antibody with isotype control immunoglobulin (R565) or antibody diluent alone (negative buffer control) during the immunohistochemical staining procedure.

- Positive controls were obtained by using AMHR2-transfected COV434 cells and human granulosa cell tumor samples.

-After processing, the sections were digitally visualized via Philips IMS. All samples were scored independently by 2 pathologists.

- Describe in detail the localization of the marker: cytoplasmic and/or membrane.

- The intensity was classified as a clear brown labeling of the tumor cell membrane and/or cytoplasm by the following scoring system: the intensity of labeling was defined as: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong, as shown by the COV434 positive control).

- Frequency was defined as the percentage of AMHRII expressing cells. Necrotic areas were excluded from the analysis. An overall histological score was established by accumulating membrane and cytoplasmic expression using the frequency x intensity score mean (0 to 3).

- Keep all slides in a safe place.

B. Results

Table 3 also depicts the results of AMHRII membrane expression of various primary human lung cancer cells, where the AMHRII expression scores represent a group of different lung cancer samples.

Table 3: AMHRII expression in human lung cancer tissue samples

Tumor type The percentage of positive samples Number of test samples SCLC 0% 2 NSCLC (neuroendocrine) 1.2% 78 NSCLC (acinar type) 0% 2 NSCLC (epidermoid) 100% 4 NSCLC (squamous cell carcinoma) 35% 14 NSCLC (adenocarcinoma) 45.8% twenty four NSCLC (large cell) 33% 9

The results indicate that AMHRII is expressed at the cell surface in a number of human lung cancer samples, in particular NSCLC-derived tumor samples, more specifically tumor samples derived from patients with NSCLC selected from the group consisting of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, and neuroendocrine NSCLC.

Example 3: AMHRII expression in lung cancer

A. Materials and Methods

A.1. Objectives

Studies were initiated with human lung cancer cells derived from patient-derived xenografts (PDX) or from fresh human tumor samples for detection of anti-Mullerian hormone type 2 receptor (AMHR2) expression using biotinylated 3C23K monoclonal antibody.

A.2. Analysis of AMHRII membrane expression by flow cytometry

Preparation of cells for analysis

- Tissues were dissected within 1 h of surgery, cut into 1-mm2 fragments, and washed in RPMI containing penicillin (10%), streptomycin (10%), and gentamicin (0.1 mg/mL; Sigma-Aldrich).

- Tissue fragments were digested with collagenase and DNase (2 mg/mL; Sigma-Aldrich) at 37°C with rapid shaking for 2 to 4 hours.

- Remove mucus and large debris by filtering through a 40-lm cell strainer.

- Viable cells were obtained by Ficoll gradient centrifugation.

The QuantumTM Simply Cellular (Bangs Laboratory) was used to quantify AMHRII binding sites on resuspended tumor cells according to the manufacturer's instructions:

- Briefly, four populations of microbeads labeled with different calibrated amounts of mouse anti-human IgG specific for the Fc portion of human IgG antibodies were stained with AlexaFluor 488-conjugated anti-AMHRII 3C23K. In a FACS tube, add one drop of each vial from the kit to 50 μl of PBS 1X:

1- Bead B (blank)

2-Beads 1+3C23K-AF 10μg/mL

3-Beads 2+3C23K-AF 10μg/mL

4-Beads 3+3C23K-AF 10μg/mL

5-Beads 4+3C23K-AF 10 μg/mL (concentration can be increased to 25 μg/ml if necessary).

- Each bead population bound different amounts of AlexaFluor488-conjugated anti-AMHRII 3C23K, generating corresponding fluorescence intensities that were analyzed on a FACS Canto II cytometer (BD).

-Generate a calibration curve by plotting the mean fluorescence intensity of each bead population against its designated antibody binding capacity (ABC).

Cells are typically stained in 1.5 ml eppendorf tubes.

- All centrifugation steps were performed at 4°C.

- All incubation steps were performed at 4°C to avoid antibody internalization.

- 3.5 million cells (trypsinized COV434-MISRII or freshly dissociated tumor cells) were centrifuged at 200-300 g for 5 minutes and washed once with PBS (500 μl per tube).

- Wash with ice-cold PBS/2% FBS (200-300 g for 3 min) and resuspend in 700 μl of PBS 1X and dispense 100 μl through FACS tubes, conditions described in Table 4 below:

Table 4

- Incubate with antibody 3C23K-AF488 in PBS/1% FBS at 4°C for 30 min

- Wash twice in PBS/2% BSA (200g to 300g for 3 min)

- Wash twice in PBS (200g to 300g for 3 min)

- Add 300 μl to 400 μl of PBS and analyze on FACS as soon as possible

This protocol did not include any fixation step for extracellular staining in order to preserve membrane integrity. Therefore, only membrane AMHRII was detected.

A.3. Immunohistochemistry: protocols and methods

- Cell lines: fixed in formaldehyde-acetate alcohol (AFA) with cell block configuration.

- Human tumors: External samples were fixed in formalin and slides from Institut Curie were fixed in AFA.

- Immunohistochemistry (IHC) techniques are possible after deparaffinization of the samples and exposure at pH 9 (microwave EZ Retriever 15' at 90°C followed by cooling during 20').

- Anti-Mullerian hormone type II receptor detection revealed by immunoperoxidase technique and DAB chromogenic substrate.

- After blocking endogenous peroxidase activity, slides were incubated with diluted biotinylated primary antibodies (1/800, 8 μg/mL) at room temperature for 90 minutes. Tissue sections were then washed with PBS and incubated with avidin/biotin ABC [carrier] complex for 30 minutes. Immunoreactive signals were detected using DAB substrate solution (DAB + substrate buffer / liquid DAB + chromogen, 10 minutes incubation). Finally, sections were lightly counterstained with Mayer's hematoxylin (Lilly's modified).

- Negative controls were obtained by replacing the primary antibody with isotype control immunoglobulin (R565) or antibody diluent alone (negative buffer control) during the immunohistochemical staining procedure.

- Positive controls were obtained by using AMHR2-transfected COV434 cells and human granulosa cell tumor samples.

-After processing, the sections were digitally visualized via Philips IMS. All samples were scored independently by 2 pathologists.

- Describe in detail the localization of the marker: cytoplasmic and/or membrane.

- The intensity was classified as a clear brown labeling of the tumor cell membrane and/or cytoplasm by the following scoring system: the intensity of labeling was defined as: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong, as shown by the COV434 positive control).

- Frequency was defined as the percentage of AMHRII expressing cells. Necrotic areas were excluded from the analysis. An overall histological score was established by accumulating membrane and cytoplasmic expression using the frequency x intensity score mean (0 to 3).

- Keep all slides in a safe place.

B. Results

a) Control

- Negative controls and isotype controls were not reactive against tumor cells.

- The positive control sample (amplified COV434 AMHRII) showed diffuse immunostaining of the cells (intensity score: 3). The labeling was uniform for cytoplasmic and membrane localization (frequency score: 100%).

- Positive granulosa cell control sample showed strong immunostaining of tumor cells (intensity score: 3). Labeling was uniform for cytoplasmic and membrane localization (frequency score: 100%).

b) AMHRII expression of patient-derived xenograft (PDX) samples as assessed by IHC.

It is important to note that membrane expression of AMHR2 appears to be underestimated when samples are fixed in formalin compared to when samples are processed in AFA.

The results of AMHRII membrane expression of various human tumors xenografted in mice are depicted in Table 5, where the AMHRII expression score represents a panel of different cancer cell types.

Table 5 below summarizes some of the results for AMHRII expression in human tumor xenografts.

Table 5: AMHRII expression in human tumor xenografts

Tumor type Percentage of positive PDX Number of PDXs tested SCLC 0% 13 NSCLC (unspecified subtype) 15.4% 13 NSCLC (epidermoid) 26.9% 26 NSCLC (adenocarcinoma) 7.7% 39 NSCLC (large cell) 40% 10

The results indicate that AMHRII is expressed at the cell surface in a variety of human lung cancer xenografts, particularly in NSCLC-derived tumor samples, more specifically tumor samples derived from patients with NSCLC selected from the group consisting of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, and some yet to be identified subtypes of NSCLC.

c) AMHRII expression in patient-derived xenograft (PDX) samples as assessed by flow cytometry (FACS)

The results depicted in Figures 3A to 3E show that, regardless of the type of lung cancer considered, AMHRII is expressed at the tumor cell membrane of xenografts derived from lung cancer patients. The results depicted in Figures 3A to 3E show that membrane protein expression of AMHRII was found in squamous cell lung cancer (Figure 3A, Figure 3C, Figure 3D), large cell lung cancer (Figure 3B) and pleomorphic cell lung cancer (Figure 3E).

In addition, for the same lung cancer cells, (i) the number of AMHRII per cell and (ii) the percentage of AMHRII cancer cells were measured in the same test sample. The results are depicted in Table 6 below.

Table 6: FACS analysis of AMHRII expression in human lung cancer cells obtained from patient-derived xenografts

In Table 6, by (i) determining the average number of AMHRII proteins present at the tumor cell membrane and by (ii) determining the percentage of membrane AMHRII positive cells in the tumor sample, the AMHRII expression in each tumor sample was assessed. The left column of Table 6 shows the indication that the corresponding tumor sample is set to "positive" or "negative". Indication "positive" means that the tumor cells of lung cancer patients significantly express AMHRII at their membranes. Indication "negative" means that AMHRII is not significantly detected at the tumor cell membrane.

The results in Table 6 indicate that all tumor samples expressed membrane AMHRII, albeit at various expression levels.

d) AMHRII expression in fresh human tumor samples as assessed by flow cytometry (FACS)

The results depicted in Figures 3F and 3G indicate that AMHRII is expressed on the membranes of tumor cells derived from surgically resected human NSCLC (Figure 3G), whereas AMHRII is not expressed on the membranes of cells derived from healthy margins generated from the same patients (Figure 3F).

e) Conclusion

AMHR2 protein expression has been confirmed in lung cancer PDX models that are positive for AMHR2 transcription. These PDXs were derived from lung IC8LC10 carcinoma and lung SC131 carcinoma. The expression level was moderate but significant, characterized by a total score of 1 to 1.5. These data suggest that AMHR2 can be expressed in all but gynecological cancers.

These models could be used in the future to characterize anti-AMHR2 therapeutics.

Example 4: In vivo efficacy of anti-AMHRII antibodies against lung cancer expressing AMHRII

1. Objective Overview

To analyze the anti-tumor efficacy of Gamamab's test compound GM102 (also referred to herein as 3C23K antibody) used as a single agent or in combination with docetaxel or combination cisplatin/gemcitabine in the SC131 patient-derived non-small cell lung xenograft model developed in immunodeficient female mice.

2. Methods

Fifty-four (54) mice bearing subcutaneously grown SC131 tumors (P22.1.3/0) ranging from 62.5 mm ³ to 220.5 mm ³ were assigned to treatment when the mean and median tumor volumes reached 130.76 mm ³ and 126.00 mm ³ , respectively.

The efficacy study XTS-1526 included 6 groups of mice, 9 mice in each group:

- In group 1, vehicle was administered at 5 ml/kg, i.v. 2qwk x 3;

- In group 2, GM102 was administered at 20 mg/kg, i.v. 2qwk x 3;

- In Group 3, docetaxel was administered once slowly i.v. at 20 mg/kg on D0;

- In Group 4, GM102 was administered at 20 mg/kg, i.v. 2qwk x 1 or 2 in combination with docetaxel administered once at 20 mg/kg slowly i.v. on D0;

- In Group 5, cisplatin was given at 5 mg/kg in combination with gemcitabine given at 100 mg/kg, both i.p. qwk x 2 or 3;

- In Group 6, GM102 was administered at 20 mg/kg, i.v. 2 qwk x 1 or 2, together with a combination of cisplatin administered at 5 mg/kg and gemcitabine administered at 100 mg/kg (both i.p. qwk x 1 or 2).

In mice not included in the efficacy study, 2 groups of 8 mice each were tested:

- In Group 7, GM102 was administered at 20 mg/kg, i.v. 2qwk x 3 in combination with cisplatin administered at 5 mg/kg, i.p. qwk x 3;

- In Group 8, GM102 was administered at 20 mg/kg, i.v. 2 qwk x 3 in combination with gemcitabine administered at 100 mg/kg, i.p. qwk x 3.

During the experiment, tumor was measured, and mice were weighed 3 times per week. From 3 mice per group, fresh tumor samples were collected, and no additional dose was applied to the sample fixed with quick-frozen tissue and formalin at D28 (for the 2nd inclusion and the 3rd inclusion) or D31 (for the 1st inclusion). Quick-frozen tissue was only advanced for subsequent analysis. The sample fixed with formalin was discarded after sampling.

3. Research objectives

The experiments described in this report were designed to determine the antitumor efficacy of a test compound (coded as GM102) alone or in combination with docetaxel or the combination cisplatin/gemcitabine (Gamamab) in the SC131 patient-derived non-small cell lung xenograft model.

Test item: GM102 (also referred to as 3C23K in this article)

The anti-AMHR2 product GM102 is a humanized mAb against the anti-Müllerian hormone receptor (AMHR2), also known as the Müllerian inhibitory substance receptor II (MISRII). AMHR2 is present at the level of the internal female sex organ precursors (Müllerian ducts) during the in utero period and is restricted to the ovaries (granulosa cells) and testes (Leydig cells) during adulthood. AMHR2 is also expressed in about 65% of gynecological cancers, such as ovaries and endometrium (Bakkum JN, Gynecol Oncol, 2007; Sahli I, Biochem, 2004; Anttonen M, Lab Invest, 2011; Song JY, Int J. Oncol, 2009).

GM102 antibody has been shown to exhibit anti-tumor efficacy in a mouse xenograft model of a human tumor cell line transfected with AMHR2. It has been shown that this efficacy depends on the participation of immune effector cells that enable antibody triggering to be optimized at the tumor level. In addition, it has been shown that GM102 efficacy has a synergistic effect with carboplatin and paclitaxel (the main chemotherapeutic agents used in ovarian cancer) (Jacquet A., Cancer Res, 2012).

Human tumor xenograft models

Human tumor samples of various histological origins are obtained with informed consent from patients treated at the Cancer Center and established as transplantable xenografts in immunodeficient mice. The implanted samples are residual material from primary tumors or metastases obtained before or after treatment. These patient-derived xenograft (PDX) models can be established without prior in vitro culture and have been studied for histological, cytogenetic, genetic and other biological markers and their response to standard of care (SOC) therapies.

The SC131 tumor model was derived from skin metastases of non-small cell lung cancer with mutant EGFR (R451F) and Kras (G12V) and wild-type TP53 and PTEN.

SC131 was a low responder to both docetaxel and the cisplatin/gemcitabine combination, and a non-responder to the other agents tested (data obtained from Swiss nude mice).

From the day of implantation, the SC131 tumor model requires approximately 17 days to obtain a maximum tumor of 60 mm ³ to 200 mm ³ and 35 to 40 days to reach 2000 mm ³ .

SC131 exhibits cachectic properties.

4. Materials

4.1. Animals and maintenance conditions

Outbred athymic (nu/nu) female mice (HSD: Athymic Nude-Foxn1 ^nu ) (ENVIGO, Ghana, France) weighing 18 to 25 g were allocated to acclimate in the animal facility with ad libitum access to food and water for at least 6 days prior to manipulation (Table 7).

Table 7. Animal characteristics

4.2. Animal Welfare Statement

Permission to use animals in CERFE facilities was obtained from the Direction des Services Vétérinaires, Ministère de l'Agriculture et de la Pêche (Agreement No. B-91-228-107). Animal care and housing complied with French regulations on the protection of experimental animals.

All experiments were performed in accordance with French regulations on the protection of experimental animals and under a currently valid vertebrate animal experimentation permit issued to Guillaume Lang by the French Ministry for Agriculture and Fisheries (No. A-75-1927, date: April 15, 2012; validity: 5 years).

4.3. Animal husbandry

During the acclimation period, mice were housed in groups of up to 7 animals, and during the experimental period, they were housed in groups of up to 6 animals. Mice were housed in individually ventilated cages (IVCs) (mm 213W x 362D x 185H, Allentown, USA) with sterile and dust-free bedding cores made of polysulfone (PSU) plastic. Food and water were sterile. Animals were housed under a light-dark cycle (14-hour artificial light day-night cycle) and controlled room temperature and humidity.

Upon request, environmental conditions are monitored and data are retained in the Central Animal House Archives.

4.4. Meal and water supply

Drinking water was provided ad libitum. A complete pelleted diet (150-SP-25 Type, SAFE) was provided daily to each mouse throughout the study. Certificates of analysis for animal food and water were maintained at the CERFE site.

4.5. Animal identification

Before each experiment, all animals were weighed and identified by means of a unique ear mold numbering system.

Each cage was identified with a paper label indicating: cage number, mouse strain and number, tumor code, and experimental date.

4.6. Test compounds and formulations

PBS IX vehicle was prepared by diluting PBS IX (Sigma PBS IX, #P5493-1L, batch SLBJ2848) 1/10 in sterile deionized water. It was stored at 4°C for processing aliquots and GM102 dilutions for 30 days.

GM102 (3C23K) concentrated aliquots (batch LP01 [R18H2-LP01]) were received on July 7, 2016 (4 vials of 5 ml, 10.1 mg/ml) and stored at 4°C. On each dosing day, the stock solution was diluted in cold PBS 1X to obtain a 2 mg/ml working solution. This solution was kept on ice or at 4°C and protected from light until processing, and then the vials were kept at room temperature during the injection process. The remaining working solution after processing was discarded.

Before each dose, 10 mg/ml of docetaxel ( Sanofi, batch 6F255A - expiration date: March 2018) The stock solution must be diluted 1/5 with 0.9% NaCl to obtain a working concentration of 2 mg/ml. The stock solution is stable for one month after reconstitution at 4°C and protected from light.

A 0.5 mg/ml stock solution of Cisplatin (Cisplatin - Teva, lot 15A30MF - expiry date: January 2017) was ready to use. The solution was kept at room temperature and protected from light until the expiry date from the supplier.

40mg/ml gemcitabine ( Lilly, batch number C442937D, expiration date: February 2018) The stock solution must be diluted 1/4 with 0.9% NaCl before each administration to obtain a working concentration of 10 mg/ml. The stock solution is stable for one month after reconstitution at 4°C and protected from light.

5. Methods

5.1. Tumor transplant model induction

Tumors of the same generation were transplanted subcutaneously into 3 to 24 mice (donor mice, passage (n-1)). When these tumors reached 700 mm ³ to 2000 mm ³ , the donor mice were sacrificed by cervical dislocation, and the tumors were aseptically excised and dissected. After removing the necrotic area, the tumors were cut into fragments measuring approximately 20 mm ³ and transferred to culture medium before implantation.

Eighty-nine (89) mice were anesthetized with 100 mg/kg ketamine hydrochloride (lot 5D92-expiration date: 03-2017, Virbac) and 10 mg/kg xylazine (lot KP0AX9X, Bayer), then the skin was sterilized with chlorhexidine solution, incised at the level of the interscapular region, and a 20 mm ³ tumor fragment was placed in the subcutaneous tissue. The skin was closed with clips.

All mice from the same experiment were implanted on the same day.

5.2. Processing period

In the XTS-1526 efficacy portion, 54 mice bearing subcutaneously grown SC131 tumors (P22.1.3/0) ranging from 62.5 mm ³ to 220.5 mm ³ were assigned based on tumor volume to yield uniform mean and median tumor volumes for each treatment group. Treatments were randomized to boxes that could accommodate up to 5 mice and treatment was initiated 18 days after tumor implantation (60% inclusion rate with staggered inclusion). The study was staggered with 5 mice per group first and then 4 mice per group 2 days later. The study was terminated 31 days after treatment initiation.

Table 8

Tumor sizes were higher and more heterogeneous for the additional groups 7 and 8. Animals were included 32 days after implantation.

Table 9

5.3. Tumor measurement and animal observation

During treatment, tumor volume was assessed by measuring tumor diameter with a caliper three times per week. The formula TV (mm ³ ) = [length (mm) x width (mm) ² ]/2 was used, where length and width are the longest and shortest diameters of the tumor, respectively.

During the treatment period, all animals were weighed 3 times per week. Adverse reactions to the different treatments were determined as:

- For each measurement, relative body weight (RBW) was calculated by dividing body weight by body weight at the start of treatment.

- Subject's percent body weight loss (% BWL) = 100 - (BW _x / BW ₀ x 100), where BW _x is the BW on any day during treatment and BW ₀ is the BW on the first day of treatment.

The mice were observed daily for physical appearance, behavioral, and clinical changes.

All signs of illness and any behavioral changes or responses to treatment were recorded for each animal.

5.4.XTS-1526 Study Design

A total of 8 groups were used, as summarized in Table 9. For Groups 1 to 6, each group initially included 9 mice. For Groups 7 and 8, each group initially included 8 mice.

In Group 1, vehicle was administered at 5 ml/kg twice a week by intravenous (i.v.) route for 3 weeks.

In Group 2, GM102 was administered at 20 mg/kg twice a week by intravenous route for 3 weeks.

In Group 3, docetaxel was administered once at 20 mg/kg by intravenous route on D0.

In Group 4, GM102 was administered at 20 mg/kg by intravenous route twice weekly for 1 or 2 weeks in combination with docetaxel administered once at 20 mg/kg by intravenous route on D0.

In Group 5, cisplatin was administered at 5 mg/kg in combination with gemcitabine administered at 100 mg/kg, both by intraperitoneal (i.p.) route once a week for 2 or 3 weeks.

In Group 6, GM102 was administered at 20 mg/kg via intravenous route twice weekly for 1 or 2 weeks, together with cisplatin at 5 mg/kg and gemcitabine at 100 mg/kg in combination (both via intraperitoneal route once weekly for 1 or 2 weeks).

In Group 7, GM102 was administered at 20 mg/kg via intravenous route twice weekly for 3 weeks together with cisplatin at 5 mg/kg via intraperitoneal route once weekly for 3 weeks.

In Group 8, GM102 was administered at 20 mg/kg via the intravenous route twice weekly for 3 weeks, along with gemcitabine at 100 mg/kg via the intraperitoneal route once weekly for 3 weeks.

All treatment doses were adjusted based on body weight at each injection.

Table 10: Doses and Dosing Schedules in XTS-1526 Efficacy Studies

5.5. Actions taken in the event of weight loss or adverse events

If any side effect was observed on the day of tumor measurement and body weight monitoring (three times a week) or weight loss ≥ 15% compared to the day of inclusion, the Sponsor was notified within the shortest delay from the discovery of the side effect/problem.

Then, implement the following behavior:

- Withhold treatment for the animal; resume treatment if weight loss is <10%;

-DietGel was given to the entire group where weight loss was observed The animals were weighed daily until weight loss was <10%; if weight loss was <10%, DietGel was discontinued. Add to.

5.6. Criteria for ethical killing

Animals were sacrificed according to the following criteria:

- Consecutive 48-hour body weight loss (BWL) ≥ 20% (3 measurements) compared to the first day of treatment.

-General changes in behavior or clinical signs.

- Tumor volume ≥ 2000 mm ³ .

5.7. Endpoints/Study Termination

Only mice that met ethical standards for sacrifice were sacrificed at the appropriate time.

All experimental groups were terminated at the end of the experimental period.

The end points of the experiment are:

- 4 weeks of treatment period,

- No follow-up period.

5.8. Blood, tumor and tissue sampling

5.8.1. Tumor sampling

5.8.1.1. Tumor sampling for FFPE

- 1/2 tumors processed for FFPE: Tumors were fixed in 10% formalin for 24 hours and transferred in ethanol 70% and sent to Histalim for paraffin embedding at the following address (i.e. 17 FFPE tumor samples [from the main study]):

The exact sampling time and formalin fixation duration were recorded for each tumor sample.

During the revision 5 editing process, the sponsor decided to discard the FFPE samples.

5.8.1.2. Tumor sampling for quick freezing

- 1/2 tumors processed for snap freezing: tumors were cut into 3x3x3mm pieces and snap frozen in liquid nitrogen, then transferred to -80°C for storage (ie 17 [from the main study] + 6 [from the tolerance cohort 2 study] snap frozen tumor samples).

The exact sampling time of each tumor sample was recorded.

Data Analysis

Data processing

All raw data are recorded in suitable combined form in numbered registers, stored and processed by computer systems.

Day 0 was considered the first day of treatment. Subsequent experimental days were numbered according to this definition.

Data are expressed as mean ± standard error of the mean (m ± sem).

The mean relative body weight curve will be generated by plotting the mean RBW of each experimental group against time. The delta relative body weight (relative body weight of the treatment group compared to the relative body weight of the control group) will be used for statistical analysis.

Mean percent body weight loss (%BWL) = 100 - (mean BWx/mean BW0 x 100), where BWx is the mean BW on any day during treatment and BW0 is the mean BW on the first day of treatment.

Tumor growth curves will be generated by plotting the mean tumor volume (in ^mm3 ) for each experimental group versus time. The Δ tumor volume (relative tumor volume of the treatment group compared to the relative tumor volume of the control group) is used for statistical analysis.

Individual tumor growth delays (TGDs) were calculated as the time (in days) required for individual tumors to reach 3 to 5 times the initial tumor volume. Median growth delays per group were calculated and reported in the table.

The tumor growth delay index (TGDI) was calculated as the median growth delay of the treatment group divided by the median growth delay of the control group.

The percentage ratio between the mean tumor volume of the treatment group (T) and the mean tumor volume of the control group (C) was calculated.

Statistical analysis was performed for each measurement using the Mann-Whitney nonparametric comparison test. Each treatment group was compared to the control group.

Tumor stability (TS) was defined as the number of mice presenting a constant tumor size in at least 3 consecutive measurements.

Partial tumor regression (PR) was defined as the number of mice presenting a tumor size lower than the initial tumor size in at least 3 consecutive measurements.

Complete tumor regression (CR) was defined as the number of mice presenting tumor sizes ranging from 0 mm ³ to 13.5 mm ³ in at least 3 consecutive measurements.

Tumor-free survivors (TFS) were defined as the number of complete tumor regressions documented until the end of the group day.

6. Results

6.1. Tolerability Data, Clinical Observations

The mean percent body weight change during the treatment period is shown in FIG4 .

In this study, mice were weighed three times a week during the experiment.

In Group 1, vehicle was well tolerated at 5 ml/kg, iv 2qwk x 3, but cachectic effects of the tumor induced a maximum mean body weight loss of 8.3% on day 16 and a maximum individual body weight loss of 17.6% on day 28. No other adverse events were observed, but animals from the second inclusion were given DietGel on days 18, 21, 25, and 26 due to cachectic effects of the tumor.

In Group 2, GM102 was well tolerated at 20 mg/kg, iv 2qwk x 3, with a maximum mean weight loss of 9.8% on Day 14 and a maximum individual weight loss of 16.8% on Day 16, corresponding to the cachectic effect of the tumor observed in Control Group 1. No other adverse events were observed, but due to cachectic effects of the tumor, DietGel was given to animals from the second inclusion on Days 11, 16, 18, and from Days 21 to 27. Mouse No. 27 was found dead on day 27 without any clinical signs.

In Group 3, docetaxel, administered once at 20 mg/kg iv on D0, induced a statistically significant (p<0.01 from Day 4) maximum mean body weight loss of 17.0% on Day 16 and a maximum individual body weight loss of 23.8% on Day 19 compared to Control Group 1. No other adverse events were observed, but due to cachectic effects of the tumor, DietGel was given to the entire group from Day 7 to Day 27 (until Day 31 for animals from the first inclusion). Despite administration of DietGel, 4 mice had to be sacrificed before completion of the study.

In Group 4, GM102 at 20 mg/kg, iv2qwk x 1 or 2, combined with docetaxel at 20 mg/kg iv once on D0, induced a statistically significant (p<0.01 from Day 4) maximum mean body weight loss of 18.1% on Day 14 and a maximum individual body weight loss of 24.1% on Day 23 compared to Control Group 1. No other adverse events were observed, but DietGel was given to the entire group on Days 4 and 5, and then from Days 7 to 27 due to the cachectic effect of the tumor. Despite administration of DietGel, 5 mice had to be sacrificed before the end of the study.

In Group 5, cisplatin at 5 mg/kg in combination with gemcitabine at 100 mg/kg (both ipqwk x 2 or 3) induced a statistically significant (p<0.01 from day 2) maximum mean body weight loss of 17.5% and a maximum individual body weight loss of 30.1% on day 11 compared to Control Group 1. Due to the combined toxicity of the compound combination and the cachectic effect of tumor growth, animals from the second inclusion were given DietGel on days 2 and 3. The entire group was then given DietGel on days 4 and 7 The entire group was then given DietGel from day 9 to day 27 (For animals from the 1st inclusion, until day 31). Despite administration of DietGel, 4 mice had to be sacrificed before the end of the study and 1 mouse was found dead on day 12.

In Group 6, GM102 at 20 mg/kg, iv 2 qwk x 1 or 2, in combination with cisplatin at 5 mg/kg and gemcitabine at 100 mg/kg (both ip qwk x 1 or 2), induced a significant (p<0.001 from day 2) maximum mean body weight loss of 21.1% and a maximum individual body weight loss of 27.5% on day 11 compared to Control 1. Due to the combined toxicity of the compound combination and the cachectic effect of tumor growth, animals from the second inclusion were given DietGel on days 2 and 3. The entire group was then given DietGel from day 4 to day 27 (For animals from inclusion 1, until day 31). Despite administration of DietGel, 7 mice had to be sacrificed before the end of the study.

In an additional group 7, GM102 at 20 mg/kg, iv 2qwk x 3, combined with cisplatin at 5 mg/kg, ip qwk x 3, and in combination with the cachectic effect of tumor growth, induced a significant maximum mean body weight loss of 12.3% at day 18 and a maximum individual body weight loss of 28.9% at day 28. Due to the combined toxicity of the compound combination and the cachectic effect of tumor growth, animals were given DietGel on days 9 and 11, and then from day 13 to day 28. Despite the administration of DietGel, 2 mice had to be sacrificed before the end of the study and 1 mouse was found dead. Also, from day 8 to the end of the study, 5 of 8 mice exhibited scaling and/or dry skin.

In an additional Group 8, GM102, given at 20 mg/kg, iv 2qwk x 3, combined with gemcitabine given at 100 mg/kg, ip qwk x 3, and in combination with the cachectic effect of tumor growth, induced a significant maximum mean body weight loss of 13.4% at day 11 and a maximum individual body weight loss of 26.4% at day 28. Due to the combined toxicity of the compound combination and the cachectic effect of tumor growth, animals were given DietGel on days 2 to 4, days 7 to 9, days 11 and 12, and then days 14 to 28. Despite the administration of DietGel, 3 mice had to be sacrificed before the end of the study and 1 mouse was found dead. Also, from day 4 to the end of the study, 6 of 8 mice exhibited scaling and/or dry skin.

6.2. Anti-tumor efficacy data

Tumor growth curves (mean tumor volume over time) are shown in Figure 4. The T/C percentage values for each treatment group are presented in Table 11 and shown in Figures 5 and 6. Statistical analysis is shown in Table 12.

In this study, tumors were measured three times per week during the experimental period.

In Group 2, GM102 administered at 20 mg/kg, i.v. 2qwk x 3 did not show any anti-tumor efficacy on Day 16 (end of control group), with TGDI = 1.33 and best T/C = 74.68%.

In Group 3, docetaxel administered once at 20 mg/kg, i.v., on D0, showed strong and statistically significant (p<0.01 on D4 and then p<0.001 from D7 to D16 compared to Control Group 1 by Mann-Whitman test) antitumor efficacy with TGDI>2.71 and best T/C=11.00% on Day 16 (end of control group), with transient tumor stabilization in 7 of 9 mice and transient partial tumor regression in 2 of 9 mice observed during the treatment period.

In Group 4, GM102 administered at 20 mg/kg, i.v. 2qwk x 1 or 2 in combination with docetaxel administered once at 20 mg/kg, i.v. on D0 showed strong and statistically significant (p<0.01 at D4, then p<0.001 from D7 to D14 compared with Control Group 1 by Mann-Whitman test) antitumor efficacy with TGDI>2.71 and best T/C=11.34% on Day 16 (end of Group 4, n=6). In addition, transient tumor stabilization was observed in 6 of 9 mice and transient partial tumor regression was observed in 3 of 9 mice during the treatment period.

In Group 5, cisplatin at 5 mg/kg in combination with gemcitabine at 100 mg/kg (both i.p. qwk x 2 or 3) showed statistically significant (p<0.01 at D4 and then p<0.001 from D7 to D11 compared to Control Group 1 by Mann-Whitman test) antitumor efficacy with TGDI=2.30 and best T/C=27.16% on Day 16 (end of Group 5, n=6). In addition, transient tumor stabilization was observed in 5 of 9 mice during the treatment period.

In Group 6, GM102 at 20 mg/kg, i.v. 2qwk x 1 or 2, in combination with cisplatin at 5 mg/kg and gemcitabine at 100 mg/kg (both i.p. qwk x 1 or 2), showed statistically significant (p<0.05 at D2, then p<0.001 from D4 to D11 compared to Control Group 1 by Mann-Whitman test) antitumor efficacy on Day 11 (end of Group 6, n=7), with TGDI=1.98 and best T/C=33.71%. In addition, transient tumor stabilization was observed in 6 of 9 mice during the treatment period.

In the additional groups 7 and 8, which could not be compared with control group 1 due to higher mean tumor volumes at entry, some transient tumor stabilization was observed during treatment, in 5 of 8 mice for the combination GM102/cisplatin and in 6 of 8 mice for the combination GM102/gemcitabine.

7. Conclusion

Results and discussion

The cachexia effect in the SC131 tumor model was higher than expected and resulted in similar weight loss in both vehicle and GM102 treated groups. Therefore, it can be considered that GM102 alone was well tolerated.

On the other hand, toxicity observed in the 4 other groups was partially attributed to the standard of care docetaxel, cisplatin, and gemcitabine and induced mortality in approximately half of the mice in each group.

The GM102 antibody used alone induced 25% tumor growth inhibition, an effect that did not reach statistical significance, while the standard of care group showed strong inhibition of tumor growth. This result was surprising because the model was initially selected based on its membrane AMHRII expression (scored 1+ by IHC). However, when membrane AMHRII expression on SC131 PDX tumors was evaluated in parallel with this study, it was noted that after several passages, membrane AMHRII expression decreased (scored 0.2+; 40% of positive cells scored 0.5%). The data confirm that AMHRII expression is unstable in certain in vitro and in vivo models, and that membrane expression is critical to the anti-tumor efficacy of AMHRII.

For the same reason, no enhancement of antitumor activity was observed by combining GM102 with these standards of care.

Example 5: In vivo efficacy of anti-AMHRII antibodies against lung cancer expressing AMHRII

A. Materials and Methods

A.1. AMHRII membrane expression by tissue immunochemistry

Therefore, a method was developed to use Alexa Fluor conjugated The indirect immunofluorescence method was performed using the anti-AMHRII 3C23K antibody 488. Signal amplification was performed in two steps using rabbit anti-AF488 antibody and goat anti-rabbit antibody of 647.

Frozen tissue sections were made using a cryostat Leica CMD1950 maintained at -20°C. The frozen tissues were fixed on metal discs with OCT compound and once solidified they were fixed on disc holders. 7 μm sections were achieved and placed on Superfrost Plus slides (Menzel ) and immediately stored at -20°C.

The cryosection slides were rehydrated with PBS 1X, then fixed at -20°C for 10 min by covering them with 300 μl cold acetone (VWR Prolabo), and recovered with parafilm to ensure that the solution completely recovered all tissues. After rinsing with PBS, the slides were treated with 300 μl blocking buffer (PBS1X-BSA 2%-goat serum 10%-TritonX100 0.1%) for 1 hour in a wet box at room temperature to block nonspecific interactions between antibodies and tissue components. 3C23K-AF488 or isotype control R565-AF488 diluted to 10 μg/ml in blocking buffer was applied in a wet box at room temperature for 30 min. After washing 3 times (3x10 min) with PBS1X-Triton X100 0.1%, anti-AF488 antibody (Invitrogen) (300 μl) diluted 1/500 in blocking buffer was added and incubated at room temperature for 30 min. After washing 3 times (3x10 min) with PBS 1X-Triton X100 0.1%, conjugated anti-rabbit antibody AF647 (Invitrogen) diluted 1/500 in blocking buffer (300 μl) was added and incubated for 30 min at room temperature. Washing (3x10 min) with PBS 1X-Triton X100 0.1% was achieved, followed by application of 0.5 μg/ml DAPI (Sigma Aldrich) for 10 min. After rinsing with PBS and H ₂ O, the slide sections were mounted under a coverslip (24x50 mm, Knittel Glass) with a drop (50 μl) of DAKO fluorescent mounting medium, avoiding air bubbles, and stored in the dark at 4°C until imaging.

Image acquisition was performed using a fluorescence microscope Leica DM5000B equipped with a CoolSnap EZ CCD camera controlled by Metavue software (Molecular Devices). Image post-processing was performed using ImageJ free software (http://imagej.nih.gov/ij/).

A.2. Human lung tumor xenografts

Tumor fragments were obtained from serially passaged xenografts of nude mice. After removal from donor mice, the tumors were cut into fragments (3 mm to 4 mm edge length) and placed in PBS containing 10% penicillin/streptomycin. The recipient animals were anesthetized by inhalation of isoflurane and received unilateral or bilateral tumor grafts subcutaneously in the flank.

LXFE2226 squamous non-small cell lung cancer model tumor xenografts were implanted subcutaneously, with one tumor per mouse (NMRI-Foxn1 ^nu from Charles River). The experiment consisted of two groups of mice, three of which were euthanized on day 15 for detection of membrane AMHRII expression by flow cytometry. The first group was a vehicle control group, and the second group received the investigational antibody GM102, which was administered intraperitoneally (ip) twice a week at a dose level of 20 mg/kg.

The antitumor efficacy was evaluated as the minimum T/C value by comparing the group median relative tumor volume (RTV) on the days when the best efficacy was achieved. The experiment was terminated on day 43 after a two-week observation period without drug administration.

Study Design:

B. Results

B. In vivo activity of GM 102 anti-AMHRII antibody against lung tumors

Tumor growth curves (mean tumor volume over time) are shown in Figure 7. Tumors were measured three times per week during the experiment.

The results depicted in Figures 7 and 8 show that the anti-AMHRII antibody GM102 exhibited potent anti-tumor activity in all treated xenograft animals.

Tumor growth measurements at day 28 showed that anti-AMHRII antibody GM102 caused a dramatic reduction in tumor volume (p<0.001), suggesting that anti-AMHRII antibody (i) prevented tumor growth and (ii) effectively caused lysis of tumor cells initially contained in the tumor xenografts.

Therefore, the results of Example 5 indicate that the anti-AMHRII antibody exerts a highly effective antitumor effect against lung cancer cells that actually express the AMHRII protein at their membranes, regardless of the expression level of the gene encoding AMHRII.

Sequence Listing

<110> Gama Mabus Pharmaceuticals

Institut Curie

<120> AMHRII binding compounds for preventing or treating lung cancer

<130> PR77729/KLP/CJ

<140> EP17305446.1

<141> 2017-04-14

<150> EP17305446

<151> 2017-04-14

<160> 74

<170> BiSSAP 1.3.2

<210> 1

<211> 318

<212> DNA

<213> Artificial sequence

<223> "3C_23 VL without a leader sequence" <223> "3C_23 VL without a leader sequence" <223> "3C_23 VL without a leader sequence" <223> "3C_23 VL without a leader sequence"

<223> "3C_23 VL without leader sequence" <223> "3C_23 VL without leader sequence"

<223> "3C_23 VL without leader sequence"

<223> "3C_23 VL without leader sequence"

<220>

<223> 3C_23 VL without leader sequence" <223> "3C_23 VL without leader sequence" <223>

"3C_23 VL without leader sequence" <223> "3C_23 VL without leader sequence

<220>

<223> 3C_23 VL without leader sequence" <223> "3C_23 VL without leader sequence

<220>

<223> 3C_23 VL without leader sequence

<220>

<223> 3C_23 VL without leader sequence

<220>

<221> CDS

<222> 1..318

<400> 1

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg gaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag 318

<210> 2

<211> 106

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 1: 1..318"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 1: 1..318

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 3

<211> 345

<212> DNA

<213> Artificial sequence

<223> "3C_23 VH without a leader sequence" <223> "3C_23 VH without a leader sequence" <223> "3C_23 VH without a leader sequence" <223> "3C_23 VH without a leader sequence"

<223> "3C_23 VH without leader sequence" <223> "3C_23 VH without leader sequence"

<223> "3C_23 VH without leader sequence"

<223> "3C_23 VH without leader sequence"

<220>

<223> 3C_23 VH without leader sequence" <223> "3C_23 VH without leader sequence" <223>

"3C_23 VH without leader sequence" <223> "3C_23 VH without leader sequence

<220>

<223> 3C_23 VH without leader sequence" <223> "3C_23 VH without leader sequence

<220>

<223> 3C_23 VH without leader sequence

<220>

<223> 3C_23 VH without leader sequence

<220>

<221> CDS

<222> 1..345

<400> 3

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc 345

<210> 4

<211> 115

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 3: 1..345"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 3: 1..345

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 4

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 5

<211> 318

<212> DNA

<213> Artificial sequence

<223> "3C_23K VL without a leader sequence" <223> "3C_23K VL without a leader sequence" <223> "3C_23K VL without a leader sequence" <223> "3C_23K VL without a leader sequence"

<223> "3C_23K VL without leader sequence" <223> "3C_23K VL without leader sequence"

<223> "3C_23K VL without leader sequence"

<223> "3C_23K VL without leader sequence"

<220>

<223> 3C_23K VL without leader sequence" <223> "3C_23K VL without leader sequence" <223>

"3C_23K VL without leader sequence" <223> "3C_23K VL without leader sequence

<220>

<223> 3C_23K VL without leader sequence" <223> "3C_23K VL without leader sequence

<220>

<223> 3C_23K VL without leader sequence

<220>

<223> 3C_23K VL without leader sequence

<220>

<221> CDS

<222> 1..318

<400> 5

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg aaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag 318

<210> 6

<211> 106

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 5: 1..318"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 5: 1..318

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 6

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 7

<211> 345

<212> DNA

<213> Artificial sequence

<223> "3C_23K VH without a leader sequence" <223> "3C_23K VH without a leader sequence" <223> "3C_23K VH without a leader sequence" <223> "3C_23K VH without a leader sequence"

<223> "3C_23K VH without leader sequence" <223> "3C_23K VH without leader sequence"

<223> "3C_23K VH without leader sequence"

<223> "3C_23K VH without leader sequence"

<220>

<223> 3C_23K VH without leader sequence" <223> "3C_23K VH without leader sequence" <223>

"3C_23K VH without leader sequence" <223> "3C_23K VH without leader sequence

<220>

<223> 3C_23K VH without leader sequence" <223> "3C_23K VH without leader sequence

<220>

<223> 3C_23K VH without leader sequence

<220>

<223> 3C_23K VH without leader sequence

<220>

<221> CDS

<222> 1..345

<400> 7

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc 345

<210> 8

<211> 115

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 7: 1..345"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 7: 1..345

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 8

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 9

<211> 639

<212> DNA

<213> Artificial sequence

<223> "3C_23 light chain without a leader sequence" <223> "3C_23 light chain without a leader sequence" <223> "3C_23 light chain without a leader sequence" <223> "3C_23 light chain without a leader sequence"

<223> "3C_23 light chain without leader sequence" <223> "3C_23 light chain without leader sequence"

<223> "3C_23 light chain without leader sequence"

<223> "3C_23 light chain without leader sequence"

<220>

<223> 3C_23 light chain without leader sequence" <223> "without leader sequence

3C_23 light chain" <223> "3C_23 light chain without leader sequence" <223>

"3C_23 light chain without leader sequence

<220>

<223> 3C_23 light chain without leader sequence" <223> "without leader sequence

3C_23 light chain

<220>

<223> 3C_23 light chain without leader sequence

<220>

<223> 3C_23 light chain without leader sequence

<220>

<221> CDS

<222> 1..639

<400> 9

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg gaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag cgg acc gtc gcc gca cca 336

agt gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act 384

gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa 432

gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag 480

agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc agc 528

acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc 576

tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc 624

aac agg gga gag tgt 639

<210> 10

<211> 213

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 9: 1..639"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 9: 1..639

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 11

<211> 1335

<212> DNA

<213> Artificial sequence

<223> "3C_23 heavy chain without a leader sequence" <223> "3C_23 heavy chain without a leader sequence" <223> "3C_23 heavy chain without a leader sequence" <223> "3C_23 heavy chain without a leader sequence"

<223> "3C_23 heavy chain without leader sequence" <223> "3C_23 heavy chain without leader sequence"

<223> "3C_23 heavy chain without leader sequence"

<223> "3C_23 heavy chain without leader sequence"

<220>

<223> 3C_23 heavy chain without leader sequence" <223> "3C_23 heavy chain without leader sequence

3C_23 heavy chain" <223> "3C_23 heavy chain without leader sequence" <223>

"3C_23 heavy chain without leader sequence

<220>

<223> 3C_23 heavy chain without leader sequence" <223> "3C_23 heavy chain without leader sequence

3C_23 heavy chain

<220>

<223> 3C_23 heavy chain without leader sequence

<220>

<223> 3C_23 heavy chain without leader sequence

<220>

<221> CDS

<222> 1..1335

<400> 11

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc gcc agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc 384

tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc 432

aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc 480

ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga 528

ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc 576

acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag 624

gtg gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac aca tgc 672

cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc 720

ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag 768

gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag 816

ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag 864

ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc 912

acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag 960

gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa 1008

gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc 1056

cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa 1104

ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag 1152

ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc 1200

tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag 1248

cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac 1296

cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 1335

<210> 12

<211> 445

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 11: 1..1335"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 11: 1..1335

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 12

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 13

<211> 639

<212> DNA

<213> Artificial sequence

<223> "3C_23K light chain without a leader sequence" <223> "3C_23K light chain without a leader sequence" <223> "3C_23K light chain without a leader sequence" <223> "3C_23K light chain without a leader sequence"

<223> "3C_23K light chain without leader sequence" <223> "3C_23K light chain without leader sequence"

<223> "3C_23K light chain without leader sequence"

<223> "3C_23K light chain without leader sequence"

<220>

<223> 3C_23K light chain without leader sequence" <223> "without leader sequence

3C_23K light chain" <223> "3C_23K light chain without leader sequence" <223>

"3C_23K light chain without leader sequence

<220>

<223> 3C_23K light chain without leader sequence" <223> "without leader sequence

3C_23K light chain

<220>

<223> 3C_23K light chain without leader sequence

<220>

<223> 3C_23K light chain without leader sequence

<220>

<221> CDS

<222> 1..639

<400> 13

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg aaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag cgg acc gtc gcc gca cca 336

agt gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act 384

gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa 432

gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag 480

agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc agc 528

acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc 576

tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc 624

aac agg gga gag tgt 639

<210> 14

<211> 213

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 13: 1..639"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 13: 1..639

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 15

<211> 1335

<212> DNA

<213> Artificial sequence

<223> "3C_23K heavy chain without a leader sequence" <223> "3C_23K heavy chain without a leader sequence" <223> "3C_23K heavy chain without a leader sequence" <223> "3C_23K heavy chain without a leader sequence"

<223> "3C_23K heavy chain without leader sequence" <223> "3C_23K heavy chain without leader sequence"

<223> "3C_23K heavy chain without leader sequence"

<223> "3C_23K heavy chain without leader sequence"

<220>

<223> 3C_23K heavy chain without leader sequence" <223> "3C_23K heavy chain without leader sequence

" <223> "3C_23K heavy chain without leader sequence" <223>

"3C_23K heavy chain without leader sequence

<220>

<223> 3C_23K heavy chain without leader sequence" <223> "3C_23K heavy chain without leader sequence

<220>

<223> 3C_23K heavy chain without leader sequence

<220>

<223> 3C_23K heavy chain without leader sequence

<220>

<221> CDS

<222> 1..1335

<400> 15

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc gcc agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc 384

tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc 432

aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc 480

ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga 528

ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc 576

acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag 624

gtg gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac aca tgc 672

cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc 720

ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag 768

gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag 816

ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag 864

ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc 912

acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag 960

gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa 1008

gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc 1056

cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa 1104

ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag 1152

ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc 1200

tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag 1248

cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac 1296

cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 1335

<210> 16

<211> 445

<212> PRT

<213> Artificial sequence

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs <223> Synthetic constructs

<223> "[CDS] from SEQ ID NO 15: 1..1335"

<223> Synthetic constructs <223> Synthetic constructs

<223> Synthetic constructs

<223> Synthetic constructs

<220>

<223> Synthetic constructs <223> Synthetic constructs <223> Synthetic

Construct <223> Synthetic construct

[CDS] from SEQ ID NO 15: 1..1335

<220>

<223> Synthetic constructs <223> Synthetic constructs

<220>

<223> Synthetic constructs

<220>

<223> Synthetic constructs

<400> 16

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 17

<211> 17

<212> PRT

<213> Homo sapiens

<223> "Signal Peptide" <223> Signal Peptide <223> Signal Peptide <223> Signal Peptide

<223> Signal peptide" <223> Signal peptide

<223> Signal peptide

<223> Signal peptide

<220>

<223> Signal peptide" <223> Signal peptide <223> Signal peptide <223>

Signal peptide

<220>

<223> Signal peptide" <223> Signal peptide

<220>

<223> Signal peptide

<220>

<223> Signal peptide

<400> 17

Met Leu Gly Ser Leu Gly Leu Trp Ala Leu Leu Pro Thr Ala Val Glu

1 5 10 15

Ala

<210> 18

<211> 556

<212> PRT

<213> Homo sapiens

<223> "Human AMHR-II lacking the signal peptide SEQ ID NO: 17" <223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17 <223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17 <223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17" <223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<220>

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17" <223>

Human AMHR-II lacking the signal peptide SEQ ID NO: 17 <223>

Human AMHR-II lacking the signal peptide SEQ ID NO: 17 <223>

Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<220>

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17" <223>

Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<220>

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<220>

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17

<400> 18

Pro Pro Asn Arg Arg Thr Cys Val Phe Phe Glu Ala Pro Gly Val Arg

1 5 10 15

Gly Ser Thr Lys Thr Leu Gly Glu Leu Leu Asp Thr Gly Thr Glu Leu

20 25 30

Pro Arg Ala Ile Arg Cys Leu Tyr Ser Arg Cys Cys Phe Gly Ile Trp

35 40 45

Asn Leu Thr Gln Asp Arg Ala Gln Val Glu Met Gln Gly Cys Arg Asp

50 55 60

Ser Asp Glu Pro Gly Cys Glu Ser Leu His Cys Asp Pro Ser Pro Arg

65 70 75 80

Ala His Pro Ser Pro Gly Ser Thr Leu Phe Thr Cys Ser Cys Gly Thr

85 90 95

Asp Phe Cys Asn Ala Asn Tyr Ser His Leu Pro Pro Pro Gly Ser Pro

100 105 110

Gly Thr Pro Gly Ser Gln Gly Pro Gln Ala Ala Pro Gly Glu Ser Ile

115 120 125

Trp Met Ala Leu Val Leu Leu Gly Leu Phe Leu Leu Leu Leu Leu

130 135 140

Leu Gly Ser Ile Ile Leu Ala Leu Leu Gln Arg Lys Asn Tyr Arg Val

145 150 155 160

Arg Gly Glu Pro Val Pro Glu Pro Arg Pro Asp Ser Gly Arg Asp Trp

165 170 175

Ser Val Glu Leu Gln Glu Leu Pro Glu Leu Cys Phe Ser Gln Val Ile

180 185 190

Arg Glu Gly Gly His Ala Val Val Trp Ala Gly Gln Leu Gln Gly Lys

195 200 205

Leu Val Ala Ile Lys Ala Phe Pro Pro Arg Ser Val Ala Gln Phe Gln

210 215 220

Ala Glu Arg Ala Leu Tyr Glu Leu Pro Gly Leu Gln His Asp His Ile

225 230 235 240

Val Arg Phe Ile Thr Ala Ser Arg Gly Gly Pro Gly Arg Leu Leu Ser

245 250 255

Gly Pro Leu Leu Val Leu Glu Leu His Pro Lys Gly Ser Leu Cys His

260 265 270

Tyr Leu Thr Gln Tyr Thr Ser Asp Trp Gly Ser Ser Leu Arg Met Ala

275 280 285

Leu Ser Leu Ala Gln Gly Leu Ala Phe Leu His Glu Glu Arg Trp Gln

290 295 300

Asn Gly Gln Tyr Lys Pro Gly Ile Ala His Arg Asp Leu Ser Ser Gln

305 310 315 320

Asn Val Leu Ile Arg Glu Asp Gly Ser Cys Ala Ile Gly Asp Leu Gly

325 330 335

Leu Ala Leu Val Leu Pro Gly Leu Thr Gln Pro Pro Ala Trp Thr Pro

340 345 350

Thr Gln Pro Gln Gly Pro Ala Ala Ile Met Glu Ala Gly Thr Gln Arg

355 360 365

Tyr Met Ala Pro Glu Leu Leu Asp Lys Thr Leu Asp Leu Gln Asp Trp

370 375 380

Gly Met Ala Leu Arg Arg Ala Asp Ile Tyr Ser Leu Ala Leu Leu Leu

385 390 395 400

Trp Glu Ile Leu Ser Arg Cys Pro Asp Leu Arg Pro Asp Ser Ser Pro

405 410 415

Pro Pro Phe Gln Leu Ala Tyr Glu Ala Glu Leu Gly Asn Thr Pro Thr

420 425 430

Ser Asp Glu Leu Trp Ala Leu Ala Val Gln Glu Arg Arg Arg Pro Tyr

435 440 445

Ile Pro Ser Thr Trp Arg Cys Phe Ala Thr Asp Pro Asp Gly Leu Arg

450 455 460

Glu Leu Leu Glu Asp Cys Trp Asp Ala Asp Pro Glu Ala Arg Leu Thr

465 470 475 480

Ala Glu Cys Val Gln Gln Arg Leu Ala Ala Leu Ala His Pro Gln Glu

485 490 495

Ser His Pro Phe Pro Glu Ser Cys Pro Arg Gly Cys Pro Pro Leu Cys

500 505 510

Pro Glu Asp Cys Thr Ser Ile Pro Ala Pro Thr Ile Leu Pro Cys Arg

515 520 525

Pro Gln Arg Ser Ala Cys His Phe Ser Val Gln Gln Gly Pro Cys Ser

530 535 540

Arg Asn Pro Gln Pro Ala Cys Thr Leu Ser Pro Val

545 550 555

<210> 19

<211> 115

<212> PRT

<213> Artificial sequence

<223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23

<223> 3C23K/3C23 <223> 3C23K/3C23

<223> 3C23K/3C23

<223> 3C23K/3C23

<220>

<223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23

<220>

<223> 3C23K/3C23 <223> 3C23K/3C23

<220>

<223> 3C23K/3C23

<220>

<223> 3C23K/3C23

<400> 19

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 20

<211> 115

<212> PRT

<213> Artificial sequence

<223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78

<223> 3C23KR/6B78 <223> 3C23KR/6B78

<223> 3C23KR/6B78

<223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78 <223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 21

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5B42 <223> 5B42 <223> 5B42 <223> 5B42

<223> 5B42 <223> 5B42

<223> 5B42

<223> 5B42

<220>

<223> 5B42 <223> 5B42 <223> 5B42 <223> 5B42

<220>

<223> 5B42 <223> 5B42

<220>

<223> 5B42

<220>

<223> 5B42

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Ala Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 22

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59

<223> K4D-24/6C59 <223> K4D-24/6C59

<223> K4D-24/6C59

<223> K4D-24/6C59

<220>

<223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59

<220>

<223> K4D-24/6C59 <223> K4D-24/6C59

<220>

<223> K4D-24/6C59

<220>

<223> K4D-24/6C59

<400> 22

Arg Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 23

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-20 <223> K4D-20 <223> K4D-20 <223> K4D-20

<223> K4D-20 <223> K4D-20

<223> K4D-20

<223> K4D-20

<220>

<223> K4D-20 <223> K4D-20 <223> K4D-20 <223> K4D-20

<220>

<223> K4D-20 <223> K4D-20

<220>

<223> K4D-20

<220>

<223> K4D-20

<400> 23

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 24

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4A-12 <223> K4A-12 <223> K4A-12 <223> K4A-12

<223> K4A-12 <223> K4A-12

<223> K4A-12

<223> K4A-12

<220>

<223> K4A-12 <223> K4A-12 <223> K4A-12 <223> K4A-12

<220>

<223> K4A-12 <223> K4A-12

<220>

<223> K4A-12

<220>

<223> K4A-12

<400> 24

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 25

<211> 115

<212> PRT

<213> Artificial sequence

<223> K5D05 <223> K5D05 <223> K5D05 <223> K5D05

<223> K5D05 <223> K5D05

<223> K5D05

<223> K5D05

<220>

<223> K5D05 <223> K5D05 <223> K5D05 <223> K5D05

<220>

<223> K5D05 <223> K5D05

<220>

<223> K5D05

<220>

<223> K5D05

<400> 25

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 26

<211> 115

<212> PRT

<213> Artificial sequence

<223> K5D-14 <223> K5D-14 <223> K5D-14 <223> K5D-14

<223> K5D-14 <223> K5D-14

<223> K5D-14

<223> K5D-14

<220>

<223> K5D-14 <223> K5D-14 <223> K5D-14 <223> K5D-14

<220>

<223> K5D-14 <223> K5D-14

<220>

<223> K5D-14

<220>

<223> K5D-14

<400> 26

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 27

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-123 <223> K4D-123 <223> K4D-123 <223> K4D-123

<223> K4D-123 <223> K4D-123

<223> K4D-123

<223> K4D-123

<220>

<223> K4D-123 <223> K4D-123 <223> K4D-123 <223> K4D-123

<220>

<223> K4D-123 <223> K4D-123

<220>

<223> K4D-123

<220>

<223> K4D-123

<400> 27

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 28

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-127/6C07 <223> K4D-127/6C07 <223> K4D-127/6C07 <223> K4D-127/6C07

<223> K4D-127/6C07 <223> K4D-127/6C07

<223> K4D-127/6C07

<223> K4D-127/6C07

<220>

<223> K4D-127/6C07 <223> K4D-127/6C07 <223> K4D-127/6C07 <223>

K4D-127/6C07

<220>

<223> K4D-127/6C07 <223> K4D-127/6C07

<220>

<223> K4D-127/6C07

<220>

<223> K4D-127/6C07

<400> 28

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Thr Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 29

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C14 <223> 5C14 <223> 5C14 <223> 5C14

<223> 5C14 <223> 5C14

<223> 5C14

<223> 5C14

<220>

<223> 5C14 <223> 5C14 <223> 5C14 <223> 5C14

<220>

<223> 5C14 <223> 5C14

<220>

<223> 5C14

<220>

<223> 5C14

<400> 29

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 30

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C26 <223> 5C26 <223> 5C26 <223> 5C26

<223> 5C26 <223> 5C26

<223> 5C26

<223> 5C26

<220>

<223> 5C26 <223> 5C26 <223> 5C26 <223> 5C26

<220>

<223> 5C26 <223> 5C26

<220>

<223> 5C26

<220>

<223> 5C26

<400> 30

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Met Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 31

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C27 <223> 5C27 <223> 5C27 <223> 5C27

<223> 5C27 <223> 5C27

<223> 5C27

<223> 5C27

<220>

<223> 5C27 <223> 5C27 <223> 5C27 <223> 5C27

<220>

<223> 5C27 <223> 5C27

<220>

<223> 5C27

<220>

<223> 5C27

<400> 31

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Pro Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 32

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C60 <223> 5C60 <223> 5C60 <223> 5C60

<223> 5C60 <223> 5C60

<223> 5C60

<223> 5C60

<220>

<223> 5C60 <223> 5C60 <223> 5C60 <223> 5C60

<220>

<223> 5C60 <223> 5C60

<220>

<223> 5C60

<220>

<223> 5C60

<400> 32

Gln Val Arg Leu Val Gln Ser Gly Ala Lys Val Arg Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 33

<211> 115

<212> PRT

<213> Artificial sequence

<223> 6C13 <223> 6C13 <223> 6C13 <223> 6C13

<223> 6C13 <223> 6C13

<223> 6C13

<223> 6C13

<220>

<223> 6C13 <223> 6C13 <223> 6C13 <223> 6C13

<220>

<223> 6C13 <223> 6C13

<220>

<223> 6C13

<220>

<223> 6C13

<400> 33

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Glu Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 34

<211> 115

<212> PRT

<213> Artificial sequence

<223> 6C18 <223> 6C18 <223> 6C18 <223> 6C18

<223> 6C18 <223> 6C18

<223> 6C18

<223> 6C18

<220>

<223> 6C18 <223> 6C18 <223> 6C18 <223> 6C18

<220>

<223> 6C18 <223> 6C18

<220>

<223> 6C18

<220>

<223> 6C18

<400> 34

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 35

<211> 115

<212> PRT

<213> Artificial sequence

<223> 6C54 <223> 6C54 <223> 6C54 <223> 6C54

<223> 6C54 <223> 6C54

<223> 6C54

<223> 6C54

<220>

<223> 6C54 <223> 6C54 <223> 6C54 <223> 6C54

<220>

<223> 6C54 <223> 6C54

<220>

<223> 6C54

<220>

<223> 6C54

<400> 35

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 36

<211> 106

<212> PRT

<213> Artificial sequence

<223> 3C23K <223> 3C23K <223> 3C23K <223> 3C23K

<223> 3C23K <223> 3C23K

<223> 3C23K

<223> 3C23K

<220>

<223> 3C23K <223> 3C23K <223> 3C23K <223> 3C23K

<220>

<223> 3C23K <223> 3C23K

<220>

<223> 3C23K

<220>

<223> 3C23K

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-K55E <223> L-K55E <223> L-K55E <223> L-K55E

<223> L-K55E <223> L-K55E

<223> L-K55E

<223> L-K55E

<220>

<223> L-K55E <223> L-K55E <223> L-K55E <223> L-K55E

<220>

<223> L-K55E <223> L-K55E

<220>

<223> L-K55E

<220>

<223> L-K55E

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 38

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T48I, L-P50S <223> L-T48I, L-P50S <223> L-T48I, L-P50S <223>L-T48I, L-P50S

<223> L-T48I, L-P50S <223> L-T48I, L-P50S

<223> L-T48I, L-P50S

<223> L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S <223> L-T48I, L-P50S <223> L-T48I, L-P50S <223>

L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S <223> L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 106

<212> PRT

<213> Artificial sequence

<223> LT48I, L-K55E <223> LT48I, L-K55E <223> LT48I, L-K55E <223>LT48I, L-K55E

<223> LT48I, L-K55E <223> LT48I, L-K55E

<223> LT48I, L-K55E

<223> LT48I, L-K55E

<220>

<223> LT48I, L-K55E <223> LT48I, L-K55E <223> LT48I, L-K55E <223>

LT48I, L-K55E

<220>

<223> LT48I, L-K55E <223> LT48I, L-K55E

<220>

<223> LT48I, L-K55E

<220>

<223> LT48I, L-K55E

<400> 39

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 106

<212> PRT

<213> Artificial sequence

<223> LS27P, L-S28P <223> LS27P, L-S28P <223> LS27P, L-S28P <223>LS27P, L-S28P

<223> LS27P, L-S28P <223> LS27P, L-S28P

<223> LS27P, L-S28P

<223> LS27P, L-S28P

<220>

<223> LS27P, L-S28P <223> LS27P, L-S28P <223> LS27P, L-S28P <223>

LS27P, L-S28P

<220>

<223> LS27P, L-S28P <223> LS27P, L-S28P

<220>

<223> LS27P, L-S28P

<220>

<223> LS27P, L-S28P

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Pro Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-T20A <223> L-M4L, L-T20A <223> L-M4L, L-T20A <223> L-M4L, L-T20A

<223> L-M4L, L-T20A <223> L-M4L, L-T20A

<223> L-M4L, L-T20A

<223> L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A <223> L-M4L, L-T20A <223> L-M4L, L-T20A <223>

L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A <223> L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A

<400> 41

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-S27P <223> L-S27P <223> L-S27P <223> L-S27P

<223> L-S27P <223> L-S27P

<223> L-S27P

<223> L-S27P

<220>

<223> L-S27P <223> L-S27P <223> L-S27P <223> L-S27P

<220>

<223> L-S27P <223> L-S27P

<220>

<223> L-S27P

<220>

<223> L-S27P

<400> 42

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 43

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W <223> L-M4L, L -S9P, L-R31W

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W

<223> L-M4L, L-S9P, L-R31W

<223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W <223> L-M4L,

L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W

<400> 43

Asp Ile Gln Leu Thr Gln Ser Pro Pro Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Trp Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 44

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L <223> L-M4L <223> L-M4L <223> L-M4L

<223> L-M4L <223> L-M4L

<223> L-M4L

<223> L-M4L

<220>

<223> L-M4L <223> L-M4L <223> L-M4L <223> L-M4L

<220>

<223> L-M4L <223> L-M4L

<220>

<223> L-M4L

<220>

<223> L-M4L

<400> 44

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I33T <223> L-I33T <223> L-I33T <223> L-I33T

<223> L-I33T <223> L-I33T

<223> L-I33T

<223> L-I33T

<220>

<223> L-I33T <223> L-I33T <223> L-I33T <223> L-I33T

<220>

<223> L-I33T <223> L-I33T

<220>

<223> L-I33T

<220>

<223> L-I33T

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Thr

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-K39E <223> L-M4L, L-K39E <223> L-M4L, L-K39E <223> L-M4L, L-K39E

<223> L-M4L, L-K39E <223> L-M4L, L-K39E

<223> L-M4L, L-K39E

<223> L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E <223> L-M4L, L-K39E <223> L-M4L, L-K39E <223>

L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E <223> L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E

<400> 46

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Glu Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T22P <223> L-T22P <223> L-T22P <223> L-T22P

<223> L-T22P <223> L-T22P

<223> L-T22P

<223> L-T22P

<220>

<223> L-T22P <223> L-T22P <223> L-T22P <223> L-T22P

<220>

<223> L-T22P <223> L-T22P

<220>

<223> L-T22P

<220>

<223> L-T22P

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 48

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-Y32D <223> L-Y32D <223> L-Y32D <223> L-Y32D

<223> L-Y32D <223> L-Y32D

<223> L-Y32D

<223> L-Y32D

<220>

<223> L-Y32D <223> L-Y32D <223> L-Y32D <223> L-Y32D

<220>

<223> L-Y32D <223> L-Y32D

<220>

<223> L-Y32D

<220>

<223> L-Y32D

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Asp Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-Q37H <223> L-Q37H <223> L-Q37H <223> L-Q37H

<223> L-Q37H <223> L-Q37H

<223> L-Q37H

<223> L-Q37H

<220>

<223> L-Q37H <223> L-Q37H <223> L-Q37H <223> L-Q37H

<220>

<223> L-Q37H <223> L-Q37H

<220>

<223> L-Q37H

<220>

<223> L-Q37H

<400> 49

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr His Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 50

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-G97S <223> L-G97S <223> L-G97S <223> L-G97S

<223> L-G97S <223> L-G97S

<223> L-G97S

<223> L-G97S

<220>

<223> L-G97S <223> L-G97S <223> L-G97S <223> L-G97S

<220>

<223> L-G97S <223> L-G97S

<220>

<223> L-G97S

<220>

<223> L-G97S

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Ser Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 51

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-S12P <223> L-S12P <223> L-S12P <223> L-S12P

<223> L-S12P <223> L-S12P

<223> L-S12P

<223> L-S12P

<220>

<223> L-S12P <223> L-S12P <223> L-S12P <223> L-S12P

<220>

<223> L-S12P <223> L-S12P

<220>

<223> L-S12P

<220>

<223> L-S12P

<400> 51

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Pro Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 52

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-19A <223> L-19A <223> L-19A <223> L-19A

<223> L-19A <223> L-19A

<223> L-19A

<223> L-19A

<220>

<223> L-19A <223> L-19A <223> L-19A <223> L-19A

<220>

<223> L-19A <223> L-19A

<220>

<223> L-19A

<220>

<223> L-19A

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 53

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T72A <223> L-T72A <223> L-T72A <223> L-T72A

<223> L-T72A <223> L-T72A

<223> L-T72A

<223> L-T72A

<220>

<223> L-T72A <223> L-T72A <223> L-T72A <223> L-T72A

<220>

<223> L-T72A <223> L-T72A

<220>

<223> L-T72A

<220>

<223> L-T72A

<400> 53

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Ala Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 54

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-R31W <223> L-R31W <223> L-R31W <223> L-R31W

<223> L-R31W <223> L-R31W

<223> L-R31W

<223> L-R31W

<220>

<223> L-R31W <223> L-R31W <223> L-R31W <223> L-R31W

<220>

<223> L-R31W <223> L-R31W

<220>

<223> L-R31W

<220>

<223> L-R31W

<400> 54

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Trp Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 55

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-M39K <223> L-M4L, L-M39K <223> L-M4L, L-M39K <223> L-M4L, L-M39K

<223> L-M4L, L-M39K <223> L-M4L, L-M39K

<223> L-M4L, L-M39K

<223> L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K <223> L-M4L, L-M39K <223> L-M4L, L-M39K <223>

L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K <223> L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K

<400> 55

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Met Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 56

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I2N <223> L-I2N <223> L-I2N <223> L-I2N

<223> L-I2N <223> L-I2N

<223> L-I2N

<223> L-I2N

<220>

<223> L-I2N <223> L-I2N <223> L-I2N <223> L-I2N

<220>

<223> L-I2N <223> L-I2N

<220>

<223> L-I2N

<220>

<223> L-I2N

<400> 56

Asp Asn Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-G63C, L-W91C <223> L-G63C, L-W91C <223> L-G63C, L-W91C <223>L-G63C, L-W91C

<223> L-G63C, L-W91C <223> L-G63C, L-W91C

<223> L-G63C, L-W91C

<223> L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C <223> L-G63C, L-W91C <223> L-G63C, L-W91C <223>

L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C <223> L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C

<400> 57

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Cys Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Cys Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 58

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-R31G <223> L-R31G <223> L-R31G <223> L-R31G

<223> L-R31G <223> L-R31G

<223> L-R31G

<223> L-R31G

<220>

<223> L-R31G <223> L-R31G <223> L-R31G <223> L-R31G

<220>

<223> L-R31G <223> L-R31G

<220>

<223> L-R31G

<220>

<223> L-R31G

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Gly Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 59

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I75F <223> L-I75F <223> L-I75F <223> L-I75F

<223> L-I75F <223> L-I75F

<223> L-I75F

<223> L-I75F

<220>

<223> L-I75F <223> L-I75F <223> L-I75F <223> L-I75F

<220>

<223> L-I75F <223> L-I75F

<220>

<223> L-I75F

<220>

<223> L-I75F

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Phe Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I2T <223> L-I2T <223> L-I2T <223> L-I2T

<223> L-I2T <223> L-I2T

<223> L-I2T

<223> L-I2T

<220>

<223> L-I2T <223> L-I2T <223> L-I2T <223> L-I2T

<220>

<223> L-I2T <223> L-I2T

<220>

<223> L-I2T

<220>

<223> L-I2T

<400> 60

Asp Thr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I2T, L-K42R <223> L-I2T, L-K42R <223> L-I2T, L-K42R <223> L-I2T, L-K42R

<223> L-I2T, L-K42R <223> L-I2T, L-K42R

<223> L-I2T, L-K42R

<223> L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R <223> L-I2T, L-K42R <223> L-I2T, L-K42R <223>

L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R <223> L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R

<400> 61

Asp Thr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 62

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-Y49H <223> L-Y49H <223> L-Y49H <223> L-Y49H

<223> L-Y49H <223> L-Y49H

<223> L-Y49H

<223> L-Y49H

<220>

<223> L-Y49H <223> L-Y49H <223> L-Y49H <223> L-Y49H

<220>

<223> L-Y49H <223> L-Y49H

<220>

<223> L-Y49H

<220>

<223> L-Y49H

<400> 62

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr His

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 63

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E <223> L-M4L, L -T20S, L-K39E

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E

<223> L-M4L, L-T20S, L-K39E

<223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E <223> L-M4L,

L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E

<400> 63

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Glu Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 64

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T69P <223> L-T69P <223> L-T69P <223> L-T69P

<223> L-T69P <223> L-T69P

<223> L-T69P

<223> L-T69P

<220>

<223> L-T69P <223> L-T69P <223> L-T69P <223> L-T69P

<220>

<223> L-T69P <223> L-T69P

<220>

<223> L-T69P

<220>

<223> L-T69P

<400> 64

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Pro Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 65

<211> 10

<212> PRT

<213> Artificial sequence

<223> "Anti-AMHRII antibody CDRL-1"

<223> Anti-AMHRII antibody CDRL-1

<220>

<223> Anti-AMHRII antibody CDRL-1

<220>

<223> Anti-AMHRII antibody CDRL-1

<220>

<221> Variants

<222> 4

<223> Xaa at position 4 is S or P

<220>

<221> Variants

<222> 5

<223> Xaa at position 5 is S or P

<220>

<221> Variants

<222> 7

<223> Xaa at position 7 is R or W or G

<220>

<221> Variants

<222> 8

<223> Xaa at position 8 is T or D

<220>

<221> Variants

<222> 9

<223> Xaa at position 9 is I or T

<400> 65

Arg Ala Ser Xaa Xaa Val Xaa Xaa Xaa Ala

1 5 10

<210> 66

<211> 7

<212> PRT

<213> Artificial sequence

<223> "Anti-AMHRII antibody CDRL-2"

<223> Anti-AMHRII antibody CDRL-2

<220>

<223> Anti-AMHRII antibody CDRL-2

<220>

<223> Anti-AMHRII antibody CDRL-2

<220>

<221> Variants

<222> 6

<223> Xaa at position 6 is K or E

<400> 66

Pro Thr Ser Ser Leu Xaa Ser

1 5

<210> 67

<211> 9

<212> PRT

<213> Artificial sequence

<223> "Anti-AMHRII antibody CDRL-3"

<223> Anti-AMHRII antibody CDRL-3

<220>

<223> Anti-AMHRII antibody CDRL-3

<220>

<223> Anti-AMHRII antibody CDRL-3

<400> 67

Leu Gln Trp Ser Ser Tyr Pro Trp Thr

1 5

<210> 68

<211> 13

<212> PRT

<213> Artificial sequence

<223> "Anti-AMHRII antibody CDRH-1"

<223> Anti-AMHRII Antibody CDRH-1

<220>

<223> Anti-AMHRII Antibody CDRH-1

<220>

<223> Anti-AMHRII Antibody CDRH-1

<220>

<221> Variants

<222> 6

<223> Xaa at position 6 is S or T

<220>

<221> Variants

<222> 9

<223> Xaa at position 9 is S or G

<220>

<221> Variants

<222> 10

<223> Xaa at position 10 is Y or N

<400> 68

Lys Ala Ser Gly Tyr Xaa Phe Thr Xaa Xaa His Ile His

1 5 10

<210> 69

<211> 17

<212> PRT

<213> Artificial sequence

<223> "Anti-AMHRII Antibody CDRH-2"

<223> Anti-AMHRII Antibody CDRH-2

<220>

<223> Anti-AMHRII Antibody CDRH-2

<220>

<223> Anti-AMHRII Antibody CDRH-2

<220>

<221> Variants

<222> 5

<223> Xaa at position 5 is G or E

<400> 69

Trp Ile Tyr Pro Xaa Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe Gln

1 5 10 15

Gly

<210> 70

<211> 6

<212> PRT

<213> Artificial sequence

<223> "Anti-AMHRII Antibody CDRH-3"

<223> Anti-AMHRII Antibody CDRH-3

<220>

<223> Anti-AMHRII Antibody CDRH-3

<220>

<223> Anti-AMHRII Antibody CDRH-3

<400> 70

Gly Asp Arg Phe Ala Tyr

1 5

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence

<223> "AMHR2 forward primer"

<223> "AMHR2 forward primer"

<220>

<223> Forward primer for AMHR2

<220>

<223> Forward primer for AMHR2

<400> 71

tctggatggc actggtgctg 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequence

<223> "Reverse primer for AMHR2"

<223> "Reverse primer for AMHR2"

<220>

<223> Reverse primer for AMHR2

<220>

<223> Reverse primer for AMHR2

<400> 72

agcagggcca agatgatgct 20

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<223> "TBP forward primer"

<223> "TBP forward primer"

<220>

<223> TBP forward primer

<220>

<223> TBP forward primer

<400> 73

tgcacaggag ccaagagtga a 21

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence

<223> "TBP reverse primer"

<223> "TBP reverse primer"

<220>

<223> Reverse primer for TBP

<220>

<223> Reverse primer for TBP

<400> 74

cacatcacag ctccccacca 20

Claims (6)

1. Use of a monoclonal antibody in the preparation of a medicament for preventing or treating lung cancer in a patient suffering from lung cancer selected from the group consisting of epidermoid non-small cell lung cancer, adenocarcinoma non-small cell lung cancer, large cell non-small cell lung cancer, squamous cell carcinoma non-small cell lung cancer, pleomorphic cell carcinoma non-small cell lung cancer and neuroendocrine non-small cell lung cancer, wherein the cancer cells of the lung cancer express human AMHRII on their membranes, wherein the monoclonal antibody comprises VL and VH, wherein: VL comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO:6; and, VH comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VH of the amino acid sequence of SEQ ID NO:8; or The monoclonal antibody comprises VL and VH, wherein: VL comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO:2; and VH comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VH of the amino acid sequence of SEQ ID NO:4. 2. The method according to claim 1, wherein the monoclonal antibody is selected from the group consisting of: a) a monoclonal antibody comprising a light chain variable region ("VL") of SEQ ID NO: 2 and a heavy chain variable region ("VH") of SEQ ID NO: 4; b) a monoclonal antibody comprising the VL of SEQ ID NO: 6 and the VH of SEQ ID NO: 8; c) a monoclonal antibody comprising a light chain of SEQ ID NO: 10 and a heavy chain of SEQ ID NO: 12; and d) a monoclonal antibody comprising a light chain of SEQ ID NO: 14 and a heavy chain of SEQ ID NO: 16. 3. The method according to claim 1, wherein the monoclonal antibody comprises a CDR consisting of the following sequence: - CDRL-1: RASSVRYIA; - CDRL-2: PTSSLKS; - CDRL-3: LQWSSYPWT; -CDRH-1:KASGYTFTSYHIH; - CDRH-2: WIYPGDDSTKYSQKFQG; and -CDRH-3:GDRFAY. 4. The method according to claim 1, wherein the monoclonal antibody comprises a CDR consisting of the following sequence: - CDRL-1: RASSVRYIA; - CDRL-2: PTSSLES; - CDRL-3: LQWSSYPWT; -CDRH-1:KASGYTFTSYHIH; - CDRH-2: WIYPGDDSTKYSQKFQG; and -CDRH-3:GDRFAY. 5. The use according to any one of claims 1 to 4, wherein the monoclonal antibody is combined with one or more different anti-cancer agents. 6. Use of a monoclonal antibody in the preparation of a kit for determining whether an individual suffering from lung cancer is likely to respond to cancer treatment using a monoclonal antibody, wherein the lung cancer is selected from the group consisting of epidermoid non-small cell lung cancer, adenocarcinoma non-small cell lung cancer, large cell non-small cell lung cancer, squamous cell carcinoma non-small cell lung cancer, pleomorphic cell carcinoma non-small cell lung cancer, and neuroendocrine non-small cell lung cancer; wherein the cancer cells of the lung cancer express human AMHRII on their membranes, and wherein the monoclonal antibody comprises VL and VH, wherein: VL comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO:6; and, VH comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VH of the amino acid sequence of SEQ ID NO:8; or The monoclonal antibody comprises VL and VH, wherein: VL comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO:2; and VH comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 consist of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VH of the amino acid sequence of SEQ ID NO:4.